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Reference  i^itirarp 


Digitized  by  the  Internet  Archive 

in  2010  with  funding  from 

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http://www.archive.org/details/textbookofphysio1904simo 


1  '.V 


A  TEXT-BOOK 


Physiological  Chemistry 


FOR 


STUDENTS  OF  MEDICINE  AND  PHYSICIANS. 


BY 


CHARLES   E.  SIMON,  M,D., 

Of  Baltimore,  Md. 
SECOND  EDITION,  REVISED  AND  ENLARGED. 


LEA   BROTHERS  &  CO., 

PHILADELPHIA    AND    NEW    YORK. 


Entered  according  to  Act  of  Congress,  in  the  year  1904,  by 

LEA    BROTHERS   &   CO., 

In  the  Office  of  the  Librarian  of  Congress,  at  Washington.    All  rights  reserved. 


OPS  13 


<?o4 


WESTCOTT    &    THOMSON,  PRESS    OF 

ELECTROTYPERS,    PHILAOA.  WfLLIAM    J.    OORNAN.    PHILAOA 


PREFACE  TO  THE  SECOND  EDITION. 


The  call  for  a  second  edition  of  the  Physiological  Chemistry  has 
afforded  the  writer  an  opportunity  of  embodying  in  the  work  the 
results  of  many  important  papers  which  have  appeared  in  the 
current  literature  since  the  first  edition  went  to  press.  The  ad- 
vances along  certain  lines  of  chemical  research  have  been  so  rapid 
within  recent  years  as  to  render  a  complete  revision  of  many  sec- 
tions imperative.  The  chapters  on  the  Albumins,  on  the  Products 
of  Nitrogenous  Katabolisra,  on  Gastric  and  Tryptic  Digestion,, 
have  been  almost  entirely  rewritten.  Many  important  additions 
have  been  made,  and  the  book  has  been  carefully  revised 
throughout.  An  appendix  of  laboratory  exercises  has  been  added, 
with  references  to  the  text.  From  these  the  instructor  can  make 
his  selection  according;  to  the  time  and  the  facilities  which  are  at 
his  disposal.  The  methods  have  been  described  in  such  detail  that 
the  student  after  his  previous  laboratory  training  should  find  no 
difficulty  in  performing  most  of  the  experiments  with  comparatively 
little  assistance  on  the  part  of  the  instructor. 

The  writer  fully  appreciates  the  kind  reception  which  the  work 
has  received  on  the  part  of  the  profession,  and  trusts  that  the  pres- 
ent edition  also  will  meet  with  approbation.  He  is  much  indebted 
to  many  friends  for  valuable  suggestions ;  and  especially  to  Professor 
AV.  E,.  Orndorff,  of  Cornell  University,  whose  friendly  criticisms 
have  been  most  helpful.  To  Messrs.  Lea  Bros.  &  Co.  his  thanks 
are  due  for  many  acts  of  courtesy. 

C.  E.  S. 

1302  Madison  Avenue,  Baltimore,  Md., 
1904. 


PREFACE  TO  THE  FIRST  EDITION. 


In  preparing  the  present  volume  on  Physiological  Chemistry  I 
have  endeavored  to  adapt  the  book  as  much  as  possible  to  the  wants 
of  the  medical  student,  and  the  physician  who  in  the  past  has  been 
unable  to  devote  the  attention  to  the  subject  which  it  merits.  The 
work  is  intended  as  a  text-book  for  the  lecture-room  and  as  a  guide 
in  the  physiological-chemical  laboratory.  Theoretical  discussions 
have  been  avoided  as  far  as  possible,  and  it  has  been  my  aim  to 
present  ascertained  facts  as  concisely  as  appeared  consistent  with  the 
importance  of  the  problems  under  consideration.  The  various 
chemical  methods  have  been  described  with  all  due  regard  to 
necessary  detail,  but  with  the  supposition  that  the  student's  course 
in  physiological  chemistry  has  been  preceded  by  a  course  in  general 
chemistry,  such  as  is  offered  now  in  the  majority  of  our  medical 
colleges. 

The  subject-matter  has  been  arranged  in  such  a  manner  that  in 
the  first  section  of  the  work  a  general  survey  is  given  of  the  origin 
and  the  chemical  nature  of  the  three  great  classes  of  food-stuffs,  and 
also  of  the  most  important  products  of  their  decomposition ;  the 
second  section  deals  essentially  with  the  processes  of  digestion,  re- 
sorption, and  excretion ;  while  the  third  portion  of  the  work  is 
devoted  to  the  chemical  study  of  the  elementary  tissues  and  the 
various  organs  of  the  animal  body,  the  specific  products  of  their 
activity  and  their  relation  to  physiological  function.  This  arrange- 
ment has  suggested  itself  to  me  as  the  most  satisfactory  for  purposes 
of  teaching. 

References  to  literature  have  been  omitted,  as  they  did  not  appear 
to  be  necessary  in  a  work  which  is  intended  primarily  for  the 
student.     The  names  of  the  grand-masters  of  physiological  chem- 


vi  PREFACE. 

istry  and  physiology,  however,  have  been  introduced  into  the  text 
as  a  matter  of  historical  interest. 

To  my  friend,  Mr,  Charles  Glaser,  of  Baltimore,  I  wish  to 
express  my  sincere  thanks  for  many  valuable  suggestions  and  aid 
in  the  revision  of  the  manuscript.  To  ^lessrs.  Lea  Bros.  &  Co. 
I  am  indebted  for  many  acts  of  courtesy. 

1302  Madisox  Aveste, 
Baxtimoee,  ^Id.,  1901. 


CONTENTS. 


CHAPTER  I. 
INTRODUCTION. 

PAGE 

General  chemical  composition  of  living  matter 17 

Forces  at  work  in  the  living  world 17 

Character  of  chemical  changes      18 

Synthetic  processes  in  plants 18 

Oxidations  and  hydrations  in  the  animal  body 19 

Chlorophy] 20 

Chemical  nature  of  chlorophyl 21 

The  food-stuffs  of  the  plant 22 

Synthesis  of  the  carbohydrates 23 

Glucosides 25 

Mannides 25 

Synthesis  of  the  fats 26 

Synthesis  of  the  albumins 26 

CHAPTER   II. 

THE  ALBUMINS. 

Elementary  composition 28 

Reaction 28 

Solubility 29 

Crystallization 29 

Diffusion 30 

Behavior  toward  polarized  light 30 

Coagulation 30 

Denaturization 32 

Behavior  toward  neutral  salts 33 

Behavior  toward  alcohol 35 

Special  reactions  of  the  albumins 35 

Precipitation 35 

Color  reactions 36 

Structural  composition 39 

Molecular  size 48 

Classification 48 

Thk  Native  Albumins 49 

The  albumins .49 

The  gloljulins 49 

The  gluco-albumins 50 

The  keratins 52 

The  histons 52 

vii 


viii  CONTENTS. 

PAGE 

The  protamins 54 

The  nucleo-albumins 56 

The  proteids 57 

The  nucleoproteids 57 

The  nucleins 58 

The  hfemoglobins  .... 59 

The  Albuminoids 00 

The  Derived  Albumins 61 

Fibrin 61 

The  coagulated  albumins 61 

The  albuminates 61 

The  albumoses 61 

The  peptones 63 

The  protones     .  ■ 64 

CHAPTER  III. 

THE  CAEBOHYDKATES. 

The  Monosaccharides 66 

The  Hexoses 66 

Glucose 69 

Lsevulose 69 

Galactose 69 

The  Pentoses 70 

The  Disacchabides 70 

Cane-sugar 71 

Maltose 72 

Isomaltose 72 

Lactose 72 

The  Polysaccharides 72 

Starch 73 

Inulin  and  lichenin 74 

Glycogen 74 

Dextrins 74 

Celluloses 74 

CHAPTER  IV. 

THE    FATS. 

The  Fats 76 

The  Lecithins 78 

The  Cholesterins 80 

CHAPTER  V. 

THE  NITEOGENOUS  DEEIVATIVES  OF  THE  ALBUMINS. 

PAGE 

The  Di-AMiDO-AciDS  (hexon  bases) 82 

Arginin 82 

Lysin 83 

Histidin 84 

The  Mono-amido  Acids 85 


CONTENTS.  ix 

PAGE 

The  Obganic,  Non-nitrogenous  Actds 91 

The  Nucleinic  Acids , 95 

The  pyrimidin  derivatives 99 

The  purin  derivatives 101 

The  Ureids 105 

The  Kreatins 108 

The  Ptomains 109 

CHAPTER  VI. 

THE  FEEMEXTS. 

General  properties Ill 

Chemical  composition  and  genei^al  reactions 115 

Mode  of  action 116 

Classification 116 

The  proteolytic  ferments 117 

The  amylolytic  ferments 117 

The  inverting  ferments 117 

The  lipolytic  ferments 117 

The  iirases 117 

Ferments  which  transform  amido-acids  into  amides 117 

The  histozyme  of  Schmiedeberg 118 

Ferments  which  cause  the  cleavage  of  glucosides 118 

The  nucleases 118 

Ferments  which  split  ofl"  carbon  dioxide 118 

The  oxidation  fennents 118 

The  oxygenases 118 

The  peroxydases 118 

The  katalases 118 

The  coagulating  ferments 119 

Keducing  ferments 119 

CHAPTER  VII. 

THE  DIGESTIVE  FLUIDS. 

The  SAI.IVA 120 

General  characteristics v 120 

Amount 120 

Chemical  composition 121 

Ptyalin 122 

Mucin 124 

Sulphocyanides 125 

Nitrites 125 

Extractives 126 

Mineral  constituents 126 

Gases 126 

The  Gastric  Juice 126 

General  considerations 126 

Amount 126 

Chemical  composition 127 

Acidity  of  the  gastric  juice 127 


X  CONTENTS. 

PAGE 

Determination  of  the  total  acidity  of  the  gastric  contents 128 

Amount 129 

Hydrochloric  acid 129 

Origin 129 

Significance 130 

Tests  for  hydrochloric  acid 131 

Estimation 132 

Lactic  acid 134 

Tests 134 

Estimation 135 

Acetic  acid  and  butyric  acid 135 

Tests 135 

Estimation 136 

Quantitative  estimation  of  the  organic  acids 136 

The  ferments  of  the  gastric  juice  and  their  pro-enzymes 136 

Pepsin 139 

Isolation 141 

Estimation 142 

Pepsinogen 142 

Tests 143 

Estimation 143 

Chymosin  and  chymosinogen 143 

Tests 144 

Isolation 144 

Estimation 145 

Other  constituents  of  the  gastric  juice 145 

Gases 145 

The  Pancreatic  Juice 146 

General  properties 147 

Amount 147 

Specific  gravity 148 

Chemical  composition 148 

The  ferments  and  their  zymogens 149 

Trypsin 150 

Test 151 

Isolation 152 

The  amylolytic  ferment 152 

Steapsin 152 

Maltase 153 

Chymosin 153 

The  Secretion  op  the  Glands  of  Brunner 153 

The  Enteric  Juice ^^^ 

Enterokinase 156 

Erepsin 156 

Secretin  and  prosecretin 157 

The  Bile 157 

Secretion 158 

Amount 159 

General  propeilies 159 

Chemical  composition 159 


CONTENTS.  xi 

PAGE 

The  mucinous  body  of  the  bile 161 

The  biliary  acids 161 

Isolation 161 

Tests 163 

Pettenkofer's  test 163 

Physiological  test 164 

Glycocholic  acid 164 

Hyoglycocholic  acid 165 

Taurocholic  acid 166 

Hyotaurocholic  acid 166 

Chenotaurocholic  acid 166 

Cholalic  acid 167 

Hyocholalic  acid  and  chenocholalic  acid 169 

Choleic  acid 169 

Fellic  acid 169 

Lithofellic  acid 169 

Taurin 170 

Isolation 171 

Glycocoll 172 

The  bile-pigments 172 

Bilirubin 173 

Tests 175 

Isolation 176 

Biliverdin 177 

Isolation 177 

Biliprasin 178 

Bilifuscin 178 

Bilicyanin 178 

Bilipurpnrin 178 

Choletelin 178 

Bilihumin 179 

Cholesterin 179 

Tests 180 

Other  organic  constituents  of  the  bile 180 

Tbe  biliary  iron 181 

CHAPTER  VIII. 

THE  PROCESSES   OF  DIGESTION  AND  EESOEPTION. 

The  Digestion  and  Resorption  of  the  Carbohydrates 182 

The  Digestion  of  the  Albumins 185 

Digestion  of  the  native  albumins 185 

Gastric  digestion 185 

Tryptic  digestion 189 

Digestion  of  the  proteids 191 

Digestion  of  the  albuminoids 192 

Resorption  of  the  Products  of  Proteolytic  Digestion 193 

Erepsin 193 

The  Digestion  and  Resorption  of  the  Fats 196 

Autolysis 196 


xii  CONTENTS. 

CHAPTER  IX. 

ANALYSIS  OF  THE  PEODUCTS  OF  ALBUMINOUS  DIGESTION. 

PAGE 

The  Products  of  Peptic  Digestion 198 

The  Products  of  Tryptic  Digestion •    ■ 199 

Reactions  of  the  individual  albumoses 200 

Hetero-alburaose 200 

Proto-albumose 200 

Gluco-albumose 201 

Deutero-fraction  A 201 

Deutero-fraction  B 204 

Deutero-fraction  C 204 

The  End-products  of  Albuminous  Digestion 204 

Antipeptone  fraction 204 

The  raono-amido-acids 205 

Leucin 205 

Tyrosin 206 

Asparaginic  acid 208 

Glutaminic  acid 209 

Glycocoll 210 

Tryptophan 211 

Antipeptone 213 

CHAPTER  X. 

BACTERIAL  ACTION   IN   THE  INTESTINAL  TRACT, 

Indol 2ie 

Skatol 217 

Phenol 218 

Ptomains 219 

Bacterial  Decomposition  of  the  Fats 219 

Bacterial  Decomposition  of  the  Biliary  Constituents 220 

CHAPTER  XI. 

THE   FECES. 

Consistence  and  form 222 

Amount 222 

Odor 222 

Color -222 

Macroscopical  constituents 223 

Microscopical  constituents 223 

Reactions      223 

General  chemical  composition 223 

Analysis  of  the  Products  of  Albuminous  Putrefaction 224 

Hydrobilirubin 225 

Excretin 226 

Stercorin 226 

Meconium 226 


CONTENTS.  xiii 

CHAPTER  XII. 

THE   URINE.  PAGE 

227 
Geneeal  Characteristics .••••• 

General  appearance ^" 

Color 229 

Odor 229 

Amount .^ 

Specific  gravity 239 

Reaction 

Determination  of  the  acidity  of  the  urine ^^'^ 

Chemical  composition " 

The  Inorganic  Constituents  of  the  Urine -o4 

Quantitative  estimation  of  the  mineral  ash ^^7 

Quantitative  estimation  of  the  chlorides 237 

Quantitative  estimation  of  the  phosphates        238 


Separate  estimation  of  the  earthy  and  alkaline  phosphates 238 

Quantitative  estimation  of  the  sulphates 


238 

Test  for  nitrates 

The  Organic  Constituents  of  the  Urine ^^ 

The  nitrogenous  constituents  of  the  urine ^ 

u^^^;.-. :;:.■;:;::'.  240 

Origin 

Nitrogenous  equilibruim ^ 

Properties 245 

Urea-nitrate ' 

Urea-oxalate 

Synthetic  formation 

Isolation 

Quantitative  estimation 

Estimation  of  the  preformed  ammonia ^'^^ 

Estimation  of  the  total  urinary  nitrogen '        JZ 

Uric  acid 252 

Origin ;    •    265 

Properties 255 

Tests o„„ 

^    ,    .  2o6 

Isolation „_^ 

.      .  ^-      r  2o6 

Quantitative  estimation 

The  xanthin-bases 

0"g'"  •.•••.•    ■. '.'.'.'.".    260 

Quantitative  estimation 

Oxalic  acid  and  oxaluric  acid ^ 

Quantitative  estimation  of  oxalic  acid 

AUantoin. ' ^g„ 

Isolation 2«o 

Kreatinin ^„. 

Properties ^    .  265 

'^^^'■^ ....    265 

Synthesis _ 

Isolation  and  quantitative  estimation 


XIV  CONTENTS. 

•  PAGE 

The  Aromatic  Constituents  of  the  Urine 266 

The  conjugate  sulphates 267 

The  plienols 267 

Quantitative  estimation 268 

Indoxyl  sulpliate 269 

Tests 271 

Quantitative  estimation 272 

Skatoxyl  sulphate 272 

Tests 273 

The  conjugate  glucuronates 274 

The  compound  glycocoUs 276 

Hippuric  acid 276 

Properties 277 

Syntliesis 278 

Isolation 278 

Quantitative  estimation 278 

Phenaceturic  acid 279 

Properties 279 

Isolation 279 

Ornithuric  acid 279 

The  Aromatic  Oxy-acids 279 

Homogentisinic  acid 281 

Inosit 282 

Kynurenic  acid 283 

The  Fatty  Acids 284 

The  volatile  fatty  acids 284 

Isolation  and  quantitative  estimation 284 

/3-oxybutyric  acid 285 

Test 286 

Estimation 286 

Diacetic  acid 287 

Tests 287 

Acetone 288- 

Tests 289 

Quantitative  estimation 290 

Lactic  acid 290 

Isolation 290 

Leucin  and  tyrosin 291 

Isolation 292 

The  Neutral,  Sitlphur  Bodies  of  the  Urine 292 

Cystein 293 

Cystin 293 

Properties 294 

Isolation  and  estimation 295 

Pre[)aration  of 296 

Estimation  of  neutral  sulphur 296 

The  C.\RBonYDRATES 297 

Glucose 297 

Tests 299 

Quantitative  estimation 302 


CONTENTS.  XV 

PAGE 

Lactose 304 

Isolation 304 

Lsevulose 305 

Laiose 305 

Maltose 305 

Dextrin 305 

Pentoses , 306 

The  Albumins 307 

Tests 308 

Estimation 313 

The  Pigments  of  the  Urine 313 

Urochrome 313 

Isolation 314 

Uroerythrin 315 

Isolation 315 

Urobilin 315 

Tests 315 

Ehrlich's  reaction 316 

The  blood-pigments 317 

Hsematin 317 

Hsematoporphyrin 317 

Urorubrohsematin  and  urofuscohsematin 318 

Melanins 319 

The  bile-pigments  .    .        320 

The  bile-acids 320 

Fats,  cholesterin,  and  lecithins 320 

Ferments 321 

Gases 321 

Ptomains 322 


CHAPTER  XIII. 

THE  ANIMAL  CELL. 

Protoplasm  and  nucleus 324 


CHAPTER  XIV. 

THE   BLOOD. 

General  considerations 329 

Physical  Characteristics  of  the  Blood 330 

Color 330 

Odor .331 

Taste 331 

Specific  gravity 331 

Amount 332 

Chemical  Examination  of  the  Blood 332 

Reaction 332 

Chemical  composition  of  the  blood  as  a  whole 334 


xvi  '  CONTENTS. 

PAGE 

The  plasma 336 

Fibrinogen 337 

Isolation 337 

Properties 337 

Serum-globulin 337 

Isolation 338 

Properties 339 

Serum-albumin 33S 

Separation  of  the  albumins  from  each  other 340 

Quantitative  estimation  of  the  albumins 340 

Serum 341 

The  coagulation  of  the  blood 342 

The  fibrin  ferment      342 

Isolation 344 

Properties 344 

Fibrin 345 

Estimation 346 

Rapidity  of  coagulation 347 

Glycogen 348 

Fat 348 

Urea 348 

The  leucocytes 349 

Xucleohiston 349 

Isolation 349 

Properties 349 

Chemical  composition 350 

The  plaques 351 

The  red  corpuscles 352 

Isolation  . 352 

Haemoglobin  and  its  derivatives 352 

Haemoglobin 352 

Globin 354 

Hsemochromogen 354 

Oxyhjemoglobin 355 

Hsematin 355 

Hsmin 357 

Carbon  dioxide  haemoglobin 360 

Carbon  monoxide  haemoglobin 360 

Nitric  oxide  haemoglobin      360 

Cyanhaemoglobin 360 

Kathaemoglobin •    •  361 

Methaemoglobin 361 

Hamatoporphyrin 362 

Phylloporphyrin 363 

Haematoidin 363 

Haemocvanin 354 


CONTENTS.  XVI I 

CHAPTER  XV. 
THE   LYMPH. 

PAGE 

Chemical  composition ^oo 

Analyses  of  different  forms 369 

Analysis  of  pericardial  fluid 369 

Analysis  of  chyle 369 

Analysis  of  cerebrospinal  fluid 370 

Analysis  of  pleural  effiision 370 

Analysis  of  peritoneal  effusion 370 

Analysis  of  hydrocele  fluid 370 

Analysis  of  amniotic  fluid 370 

Analysis  of  pus 371 

The  Synovial  Fluid 371 

CHAPTER  XVI. 

THE  MUSCLE-TISSUE. 

Analysis  of  fresh  muscle-tissue 372 

The  Muscle-albumins 373 

Muscle-plasma 373 

Myogen 374 

Isolation 374 

Myosin 375 

Significance  of  the  common  muscle-albumins 376 

Other  albumins 377 

Myoproteid 377 

Nucleoproteids 377 

Phosphor-carnic  acid 377 

Ferments 378 

Muscle-stroma 379 

The  Muscle-pigments 379 

Glycogen 379 

Glucose 383 

Lactic  Acid 383 

Isolation  and  quantitative  estimation 386 

Inosit 387 

Tests 387 

Isolation 387 

The  Nitrogenous  Extractives 388 

Kreatin  and  kreatinin 388 

Properties  of  kreatin 389 

Isolation  of  kreatin 390 

The  xanthin-bases 390 

Isolation 391 

Xanthin 392 

Hypoxanthin 392 

Guanin 393 

Adenin 393 


xviii  CONTENTS. 

PAGE 

Carnin 374 

Inosinic  acid 394 

Camosin 395 

Gases 395 

Fat 395 


CHAPTEE   XVII. 

THE   XERVE-TISSUE. 

Analysis 397 

Albumins 398 

Neurokeratin 399 

The  Myelin  Bodies 400 

Protagron 400 

Cerebrin 401 

Homocerebrin 402 

Encephalin 403 

Lecithins 403 

Cholesterins 404 

The  extractives 404 

Neuridin 404 

Jecorin 405 

CHAPTER   XVIII. 

THE   EYE  AND   THE   EAE. 

The  Eye 406 

The  cornea 406 

The  sclerotic 406 

The  aqueous  humor 406 

The  crystalline  lens 407 

The  vitreous  body 408 

The  retina 408 

Rhodopsin 409 

Chromophanes 410 

The  choroid 410 

The  Ear 410 

CHAPTER  XIX. 
THE  SUPPOETING  TISSUES. 

Mucous  tissue 411 

"White  fibrous  tissue 411 

Yellow  elastic  tissue 412 

Reticulated  tissue 412 

Cartilage 412 

Chondroitin-sulphuric  acid 413 

Chondromucoid 414 

Alburaoid 415 

Mineral  constituents 415 


CONTENTS.  xix 

PAGE 

BoxE 415 

Bone-marrow 417 

The  Teeth 417 

Dentin 417 

Cement 417 

Enamel 417 

Adipose  Tissue 418 

Analysis  of  adipose  tissue 420 

Origin  of  the  fats 420 

Significance  of  the  fats 422 

CHAPTER   XX. 

THE   SKIN  AND   ITS  APPENDAGES. 

The  sweat 426 

Gases 428 

The  sebum 428 

The  cerumen 429 

CHAPTER  XXI. 
THE   GLANDULAE  OKGANS  OF  THE  BODY. 

The  Liver 430 

The  albumins 431 

Ferments 434 

Glycogen " 434 

Glucose 435 

Fat 435 

Extractives 436 

The  Digestive  Glands 436 

The  Lymph-glands 437 

The  Kidneys 438 

The  Mammary'  Glands 438 

The  milk 439 

General  characteristics 439 

Amount 441 

Specific  gravity 441 

Reaction 441 

Chemical  composition 442 

The  albumins 443 

Casein 443 

Lactalbumin 445 

Lactoglobulin 446 

The  fats 447 

Lactose 448 

The  extractives 449 

Ferments 450 

Colostrum 450 

The  Reproductive  Glands 451 

The  testicles 451 

The  semen 452 


XX  CONTENTS. ' 

PAGE 

The  spermatic  liquid 453 

Spermin 453 

The  spermatozoa 454 

The  ovaries 455 

The  ovum .• 455 

The  shell 455 

The  albumen 456 

The  yolk     459 

Incubation 463 

CHAPTEE  XXII. 

THE  DUCTLESS  GLANDS. 

The  Thyroid  Gland 466 

Thyreoglobulin 467 

Thyreo-nucleo-proteid 468 

Extractives      469 

The  Adrenal  Glaxds 469 


APPEXDIX. 

Laboratory  Exercises 473 


PHYSIOLOGICAL    CHEMISTRY. 


CHAPTER    I. 

INTRODUCTION. 


The  science  of  physiological  chemistry  has  for  its  object  the 
study  of  the  various  chemical  processes  which  take  place  in  the 
bodies  of  animals  and  plants,  and  which  are  more  or  less  intimately 
associated  with  the  phenomena  of  life.  As  the  phenomena  of  life, 
moreover,  are  essentially  dependent  upon  the  transformation  of 
living  matter  into  non-living  matter,  and  vice  versa,  physiological 
chemistry  deals  primarily  with  the  chemical  processes  of  nutrition 
in  the  widest  sense  of  the  term.  Its  study  therefore  comprises  a 
consideration  of  the  various  substances  which  are  generally  desig- 
nated as  food-stuifs,  their  origin,  their  transformation  into  living 
tissue,  and  their  ultimate  fate. 

General  Composition  of  Living  Matter. — Chemical  examina- 
tion shows  that  plants  and  animals  consist  essentially  of  carbon, 
hydrogen,  nitrogen,  oxygen,  sulphur,  phosphorus,  chlorine,  potas- 
sium, sodium,  calcium,  magnesium,  and  iron — that  is,  of  elements 
which  occur  also  widely  distributed  in  the  non-organized  world. 
In  the  bodies  of  animals  and  plants  these  elements  are  built  up  to 
form  bodies  of  highly  complex  chemical  constitution,  which  belong 
to  the  class  of  albumins,  carbohydrates,  and  fats.  Upon  their  pres- 
ence both  animals  and  plants  are  dependent  for  their  existence,  and 
as  these  bodies  are  constantly  being  broken  down  and  transformed 
into  simpler  chemical  compounds,  as  the  result  of  the  various  mani- 
festations of  life,  it  follows,  from  the  law  of  the  indestructibility 
of  matter,  that  for  their  replacement  the  living  body  is  forced  to 
depend  upon  such  simpler  matter  as  is  pre-existent.  This  matter 
it  is  capable  of  transforming  into  the  complex  substances  of  which 
its  tissues  are  composed. 

Forces  at  Work  in  the  Living  World. — The  forces  which  are 
at  work  in  eifecting  these  various  changes  are  apparently  the  same 
as  those  whicli  are  operative  in  the  non-organizecl  world.  For  the 
assumption  of  special  vital  forces  there  seems  to  be  less  necessity 
the  more  we  come  to  understand  the  mechanism  of  vital  phe- 
nomena. 

2  17 


18  INTRODUCTION. 

Character  of  Chemical  Changes. — The  chemical  processes 
"vvhich  are  involved  in  the  transformation  of  non-living  matter  into 
living  tissue  are  qnalitatively  the  same  in  phmts  and  animals. 
Quantitative  dillerences,  however,  exist,  wliieh  are  sufficientlv  ])ro- 
uounced  to  serve  as  marks  of  distinction  between  animals  and 
plants.  Thus  plants  are  capable  of  evolving  from  relatively  simple 
compounds  those  complex  chemical  substances  which  go  to  form 
their  structure,  while  animals  apparently  do  not  possess  this  poAver. 
They  are  hence  dependent  for  their  existence  upon  food-stuffs  wliich 
are  preformed ;  and  the  potential  energy  which  animals  require 
for  the  functioning  of  their  various  organs,  and  which  they  trans- 
form into  kinetic  energy,  is,  as  a  matter  of  fact,  derived  in  every 
instance,  either  directly  or  indirectly,  from  plant-life.  Plants,  in 
turn,  obtain  the  potential  energy  which  is  stored  in  their  tissues  from 
the  kinetic  energy  of  sunlight,  and  in  virtue  of  this  energy  can 
elaborate  those  simple  chemical  substances  which  are  at  their  dis- 
posal as  food-stuffs  into  the  complex  bodies  which  constittite  their 
tissues. 

AVe  thus  observe  that  while  in  plant-life  synthetic  chemical 
processes  prevail,  analytical  processes  are  foremost  in  animal  life. 
These  analytical  processes,  moreover,  are  largely  of  the  character  of 
oxidations,  while  the  syntheses  which  are  effected  in  the  bodies  of 
plants  are  essentially  of  the  nature  of  reductions.  But  just  as  syn- 
thetic processes  are  not  absolutely  characteristic  of  plant-life,  so  also 
do  oxidation-processes  occur  in  plants,  and  synthetic  reductions  in 
animals.  This  becomes  especially  noticeable  as  we  descend  in  the 
scale  of  both  animal  and  vegetable  life.  Primitive  vegetable 
organisms  are  thus  met  with  which,  like  the  highly  organized  mam- 
mal, are  almost  entirely  dependent  for  their  existence  upon  already 
elaborated  food-stuffs,  and  low  forms  of  animal  life  similarly  occur 
in  which  the  processes  of  nutrition  are  essentially  the  same  as  those 
which  occur  in  the  higlier  plants.  The  differences  which  thus  exist 
between  animal  life  and  plant-life  are  therefore,  as  has  been  stated, 
more  of  a  quantitative  than  a  qualitative  kind. 

Synthetic  Processes  in  Plants. — I  have  said  that  plants  are 
capable  of  elaborating  from  simpler  compounds  the  complex  chem- 
ical substances  of  which  they  are  composed,  and  that  the  chemical 
processes  here  involved  are  essentially  of  the  nature  of  synthetic 
reductions.  Formerly,  it  was  believed  that  the  various  organic  sub- 
stances which  occur  in  animals  and  plants  could  be  produced  only 
through  the  agency  of  a  special  vital  force ;  but  A\"e  now  know  that 
this  is  not  necessarily  the  case,  and  that  as  a  matter  of  fact  a  large 
numl^er  of  such  bodies  can  be  produced  artificially  in  the  chemical 
laboratory.  AVohler,  in  1829,  was  the  first  to  demonstrate  this 
possibility  by  preparing  urea  from  ammoniinn  cyanate.  This  he 
accomplished  by  heating  the  substance  to  a  temperature  (^f  100°  C, 
when  a  transposition  of  atoms  apparently  takes  place,  and  urea 
results.     The  force   which   is  necessarv  to  effect  such  a  change  is 


OXIDATIONS  AND  HYDRATIONS  IN  THE  ANIMAL  BODY.      19 

here,  as  in  many  syntheses  M'hieh  can  artificially  be  hrouglit  about, 
a  relativ'ely  hi<2:h  temperature.  In  the  bodies  of  animals  and  plants 
a  like  temperature,  of  course,  would  destroy  life,  and  there  must 
hence  be  a  different  mechanism  at  the  disposal  of  living  beings  to 
effect  such  a  change.  We  know  that  under  the  influence  of  sun- 
light certain  plants  are  capal)le  of  effecting  the  synthesis  of  carbo- 
hydrates, fats,  and  albumins  from  the  carbon  dioxide  of  the  air, 
and  the  water  and  certain  mineral  salts  of  the  soil,  and  that  the 
ability  to  bring  about  these  changes  is  in  a  large  measure  dependent 
upon  the  presence  of  a  chemical  substance  which  is  found  in  the 
green  parts  of  plants,  and  which  is  termed  chlorophyl.  We  know 
further  that  chlorophyl  requires  exposure  to  sunlight  to  effect  these 
changes,  but  of  the  mechanism  through  which  these  changes  are 
brought  about  we  know  nothing. 

Oxidations  and  Hydrations  in  the  Animal  Body. — The  oxida- 
tion-processes which  prevail  in  animals,  and  in  consequence 
of  which  the  more  complex  substances  which  go  to  form  the 
various  tissues  and  organs  of  the  body  are  retransformed  into 
those  simple  compounds  which  plants  require  for  their  exist- 
ence, we  are  also  unable  to  explain.  We  know  that  the  oxy- 
gen of  the  air,  as  also  that  of  the  blood,  exists  in  a  neutral 
molecular  form,  and  as  such  is  incapable  of  effecting  the  oxi- 
dation of  such  complex  substances  as  the  albumins  and  fats. 
The  older  view  that  oxygen  exists  in  the  body  as  ozone,  and  that 
the  various  oxidation-processes  take  place  in  the  animal  fluids,  has 
been  abandoned,  and  it  is  now  generally  accepted  that  these  changes 
occur  in  the  individual  cells.  Here,  then,  a  splitting  up  of  the 
neutral  oxygen  must  take  place,  but  of  the  forces  which  effect 
this  decomposition  we  know  next  to  nothing.  Whether  Ave  believe 
with  Pfliiger  that  the  organized  living  albumin,  in  contradistinction 
to  the  non-organized  circulating  albumin,  is  characterized  by  a 
greater  motility  of  its  atoms,  in  consequence  of  which  the  neutral 
oxygen  is  decomposed,  or  whether  we  accept  the  view  that  reducing- 
substances  are  formed  during  the  decomposition  of  the  albuminous 
molecule  in  consequence  of  the  activity  of  a  third  factor,  we  are 
as  far  removed  from  an  adequate  explanation  of  these  phenomena 
as  in  the  beginning. 

Within  recent  years  repeated  observations  have  shown  that  from 
various  organs  of  the  body  certain  substances  can  be  extracted  which 
are  apparently  identical  with  or  closely  related  to  the  so-called  en- 
zymes. Certain  representatives  of  this  class,  such  as  pepsin,  trypsin, 
ptyalin,  and  others,  are,  as  we  shall  see,  formed  in  the  cells  of  the 
digestive  glands  of  the  l)ody,  and  serve  the  purpose  of  transforming 
the  various  food-stutts  which  are  furnished  the  animal  by  the  plant 
into  forms  which  can  be  absorbed  and  built  up  into  its  tissues.  The 
chemical  processes  which  are  here  involved  are  essentially  of  the 
character  of  hydrations.  Other  bodies,  however,  of  this  order 
which  can    be    obtained  from    living   tissues,    and    which  are  also 


20  INTRODUCTION. 

capable  of  manifesting  their  special  activity  after  the  death  of  tlieir 
parent-cells,  ajiparently  possess  the  power  of  oxidation,  and  it  is 
hence  possible  that  these  processes  in  the  living  tissues  may  also  be 
referable  to  such  enzymatic  activity.  Whether  this  is  actually  the 
case  is  not  definitely  known.  But  if  so,  we  are  apparently  approach- 
ing a  time  when  what  we  have  heretofore  been  forced  to  ascribe 
to  the  activity  of  a  special  vital  force  may  be  ex])lained  upon  the 
basis  of  jihysical  laws  which  are  seen  also  at  work  in  the  non-organ- 
ized world.  For  we  know  that  properties  which  are  sup]wsedlv 
characteristic  of  the  enzymes  are  possessed  also  by  certain  elements 
which  are  found  only  in  the  inorganic  world.  The  most  notable 
properties  of  the  enzymes  are  their  ability  to  effect  an  amount  of 
chemical  change  which  appears  to  be  out  of  all  proportion  to  the 
quantity  of  the  enzyme  present,  and  the  fact  that  the  enzyme  itself 
apparently  does  not  enter  into  the  reaction.  These  same  properties, 
however,  are  common  to  certain  metals  and  their  oxides.  Bredig 
and  von  Berneck  showed  that  a  gram-atomic  weight  (193  grams)  of 
colloidal  platinum  diffused  through  70,000,000  liters  of  water  shows 
a  perceptible  action  on  more  than  1,000,000  times  the  quantity  of 
hydrogen  peroxide  ;  and  H.  C.  Jones  demonstrated  that  the  reac- 
tion which  here  takes  place  is  a  mono-molecular  reaction,  which 
indicates  that  the  platinum  itself  does  not  enter  into  the  reac- 
tion. Curiously  enough,  the  analogy  between  the  action  of  such 
metallic  solutions  and  that  of  the  enzymes  goes  still  further.  Finely 
divided  platinum,  palladium,  iridium,  osmium,  etc.,  thus  have  the 
power  of  inverting  cane-sugar,  like  one  of  the  enzymes,  invertin  ; 
and  certain  poisons,  such  as  hydrocyanic  acid,  sulphuretted  hy- 
drogen, carbon  disulphide,  and  mercuric  chloride,  which  inhibit  or 
even  suspend  the  action  of  the  enzymes  entirely,  exert  a  similar 
influence  upon  a  solution  of  colloidal  platinum.  Without  entering 
upon  this  very  interesting  subject  further,  it  is  clear  that  a  path  has 
been  opened  upon  which  it  may  be  possible  to  penetrate  into  the 
mysteries  of  the  so-called  vital  forces,  and  to  show  ultimately  that 
such  forces  are  essentially  the  same  as  those  met  with  in  the  non- 
living world. 

Chlorophyl. — In  the  light  of  more  recent  investigation,  it  seems 
probable  that  some  of  the  synthetic  processes  which  occur  in  plant- 
life  may  also  be  referable  to  the  action  of  enzymes.  As  a  matter 
of  fact,  such  bodies  are  abundantly  present  in  the  vegetable  world, 
and  we  know  that  some  of  these  at  least,  and  probably  all,  are 
characterized  by  a  reversible  activity.  Maltase,  a  ferment,  which, 
as  we  shall  see  later,  causes  inversion  of  the  disaccharide  maltose  to 
glucose,  is  thus  similarly  able  to  bring  about  the  synthetic  formation 
of  maltose  from  two  molecules  of  glucose.  On  the  other  hand,  it 
appears  that  the  primary  formation  of  food-stuffs  in  plants  does 
not  occur  in  this  manner,  but  is  referable  to  the  activity  of  a  special 
body  which,  as  has  been  stated,  is  present  in  the  exposed  green 
parts  of  most  plants,  and  which  is  termed  chlorophyl.     This  sub- 


CHLOROPHYL.  21 

stance  occurs  widely  distributed  in  the  vegetable  world,  but  is  also 
found  in  those  low  forms  of  animal  life  in  which  the  processes  of 
nutrition  are  essentially  the  same  as  those  met  with  in  the  higher 
plants.  In  itself,  however,  chlorophyl  is  incapable  of  bringing 
about  those  syntheses  which  are  characteristic  of  vegetable  life,  and 
in  the  cells  of  the  foliage  of  plants  it  occurs  in  combination  with 
certain  albuminous  bodies,  in  the  form  of  the  so-called  chlorophylic 
granules.  These  are  apparently  special  elementary  organisms,  and 
endowed  with  a  power  of  locomotion  analogous  to  that  of  amoebae 
and  leucocytes,  so  that  they  can  approach  the  surface  of  the  leaf  to 
seek  the  sunlight,  or  retreat  when  this  becomes  too  intense.  In  a 
germinating  plant  which  has  not  been  exposed  to  sunlight  the  green 
color  is  wanting,  but  in  the  cotyledons  is  found  a  differentiation  of 
the  cellular  protoplasm  into  small  yellowish  bodies,  which  have  been 
termed  kucites.  When  these  bodies  are  exposed  to  light,  even  for  a 
relatively  short  time,  they  assume  a  green  color,  and  then  constitute 
the  chlorophylic  granules.  I  have  said  that  chlorophyl — that  is, 
the  green  coloring-matter  of  plants — is  unable  to  effect  synthetic 
changes,  and  the  same  is  true  of  the  colorless  leucites.  Chlo- 
rophyl thus  apparently  forms  an  integral  constituent  of  the  function- 
ing leucites,  and  all  those  chemical  and  physical  influences  which 
bring  about  the  destruction  of  the  protoplasm  similarly  destroy  the 
power  of  chlorophyl  to  manifest  its  special  activity.  In  the  living 
plant,  however,  it  becomes  active  at  once  upon  exposure  to  sunlight, 
and  is  then  capable  of  effecting  those  complicated  syntheses  of  which 
mention  has  been  made.  In  the  dark  it  again  becomes  inactive,  and 
the  plant  is  then  obliged  to  live  like  an  animal — by  consuming  its 
stored  energy. 

Chemical  Nature  of  Chlorophyl. — Of  the  chemical  composi- 
tion and  structure  of  chlorophyl  we  know  little  that  is  definite. 
Numerous  attempts  have  been  made  to  isolate  it  from  the  living 
plant,  but  it  is  doubtful  whether  any  of  these  attempts  has  yielded 
the  actual  substance.  Only  its  decomposition-products,  or  at  best 
very  impure  forms,  have  apparently  been  obtained.  Gautier,  it  is 
true,  claims  to  have  isolated  the  substance  in  crystalline  form  by 
methods  which  are  calculated  to  avoid  its  chemical  alteration. 
Others,  however,  have  not  been  successful  in  repeating  his  work. 

The  substance  which  Gautier  obtained  from  spinach-leaves  oc- 
curred in  the  form  of  small  crystals  of  a  dark-green  color,  which 
on  exposure  to  light  turned  brown,  then  yellow,  and  finally  became 
colorless.  Its  composition  corresponded  to  the  formula  C^^Hg^NjO^. 
The  mineral  ash  consisted  of  about  1.75  per  cent,  of  magnesium 
phosphate,  traces  of  calcium  and  sulphates,  while  iron  was  absent. 
Treated  with  hydrochloric  acid,  it  was  decomposed  into  phylloxanthin 
and  phyllocycmic  acid,  CsJi^^^^Of^  or  C3,;H4yN^06.  This  latter  is 
thus  a  homologue  of  bilirubm,  €32113^X40^,  which  in  turn  is  derived 
from  luematin,  and  is  isomeric  with  hannatoporpJri/rin.  A  most 
interesting  relationship  between  the  blood  coloring-matter  hsemo- 


22  lyiROLUCTioy. 

globin  and  the  vegctalile  colorino-mattor  clilorophyl  thus  becomes 
apparent,  and  con^^titute.-  a  further  link  connecting  the  animal  \vith 
the  vegetable  ^vorld.  Recent  inverftigations  iiave  shown  that  a 
substance  can  be  oljtained  from  chlorophyl.  Avhich  is  termed  phvllo- 
porphyrin,  and  which  differs  from  hsemat<ipoqihyrin  anhydride 
only  in  containing  three  atoms  less  of  oxygen,  viz.,  C^Hj^X^Oj. 
Both  pliylloporphyriu  and  haematoporphyrin,  yield  the  same 
decomposition-product  on  reduction  with  phosphonium  iodide  and 
hydriodic  acid,  viz.,  hsemopyrrol,  C^H^jX  :  and  if  the  reduction  of 
hsematopoq^hyrin  is  not  carried  on  too  energeticallv  mesoporphvrin 
results  ( CgoHj^.X^O J,  which  manifestly  stands  midway  between 
hsematoporphyrin  and  phylloporphyrin. 

Moderately  concentrated  solutions  of  clilorophyl  in  alcohol  or 
petroleum-ether  show  seven  bands  of  absorption.  The  first  of  these, 
I,  is  situated  in  the  red  portion  of  the  spectrum  between  B  and  C, 
and  is  well  pronounced  and  sharjily  defined  on  both  sides.  The 
bands  II,  III,  and  IV  are  rather  indistinct  and  scattered  through 
the  orange-yellow,  the  yellow  and  the  yellowish-green  portion  be- 
tween C  and  E.  From  F  off,  the  greater  portion  of  the  spectrum 
is  absorbed  by  the  remaining  bands,  V,  VI,  and  VII.  of  which  V  is 
seen  to  the  right  of  F,  VI  most  marked  about  G,  while  VII  occu- 
pies the  extreme  violet  end.  Very  concentrated  solutions  allow  the 
red  rays  to  pass  only  as  far  as  B.  while  in  greater  dilution  the  green 
rays  likewise  appear.  Such  solutions,  therefore,  appear  green  when 
viewed  with  transmitted  light,  while  with  reflected  light  they  are 
red  and  fluorescent. 

When  a  fresh  leaf  is  similarly  examined,  a  spectrum  is  obtained 
which  is  essentially  the  same  as  that  just  described.  There  is  lack- 
ing, however,  the  band  that  corresponds  to  the  red  fluorescent  rays 
of  chlorophyl  solutions.  This  is  explained  by  the  assumption  that 
the  red  rays  are  absorbed  by  living  chlorophyl  and  transformed  into 
chemical  energy.  In  accordance  with  this  view,  we  find  that  when 
living  plants  are  successively  ex|X)sed  to  the  various  rays  constitut- 
ing sunlight,  decomposition  of  carbon  dioxide  with  liberation  of 
oxygen — which,  as  we  shall  presently  see,  takes  place  in  the  green 
pr)rtious  of  every  plant  whenever  it  is  exposed  to  sunlight — occurs 
with  special  intensity  when  the  plant  is  exp<^»sed  to  the  rays  corre- 
sponding to  the  bands  I.  II.  and  III.  In  this  manner,  then,  chloro- 
phyl-bearing  plants  derive  their  kinetic  energy  from  sunlight,  and 
thus  become  enabled  to  elaljorate  the  simple  food-stuffs  which  are 
at  their  disposal  into  the  complex  substances  which  constitute  their 
tissues. 

The  Food-stuffs  of  Plants. — The  most  essential  elements  which 
enter  into  the  composition  <if  the  tissues  of  plants  are,  as  has  l^een 
pointed  out,  carl^)n,  hydrogen,  oxygen,  and  nitrogen.  These  sub- 
stances are  available  to  the  plant  as  carlxm  dioxide,  water,  and  cer- 
tain nitrates.  The  origin  of  the  first  mentioned  is,  of  course,  obvious, 
while  tliat  of  the  last  is  at  first  sight  somewhat  obscure. 


SYNTHESIS  OF  THE  CARBOHYDRATES.  23 

The  nitrates  are  present  in  any  soil  which  contains  organic 
matter,  and  are  now  known  to  result  from  this  through  the  special 
activity  of  certain  bacteria.  Decomposing  animal  and  vegetable 
matter  is,  however,  not  the  only  source  of  the  nitrates,  for  it  can  be 
demonstrated  that  arable  soil,  apparently  devoid  of  vegetable  life,  is 
capable,  unless  sterilized,  of  fixing  a  very  considerable  amount  of 
nitrogen,  wiiich  must  of  necessity  be  derived  from  the  atmosphere. 
The  assumption  has  been  that  this  nitrogen  is  obtained  as  an  ammo- 
nium compound,  which  the  bacteria  then  transform  into  nitrates. 
In  this  connection  it  is  interesting  to  note  that  the  hydroxides  of 
some  of  the  heavy  metals  (iron,  cobalt,  nickel)  are  capable  of  pro- 
ducing nitrites  in  minute  quantities  from  the  nitrogen  of  the  atmos- 
phere, and  that  the  first  step  in  the  formation  of  nitrates  may  hence 
be  a  purely  chemical  one.  "We  do  not  wish  to  convey  the  impression, 
however,  that  all  plants  require  their  nitrogen  in  this  form,  for  we 
know  that  Saccharomyces  cerevisiae,  for  example,  can  elaborate  its 
nitrogen  from  ammonium  salts  directly,  and  is  even  incapable  of 
utilizing  that  which  is  furnished  in  the  form  of  nitrates.  Under  cer- 
tain conditions,  moreover,  probably  all  plants  can,  for  a  time  at 
least,  grow  in  the  presence  of  ammonium  nitrogen  only. 

The  necessary  mineral  salts  the  plant  likewise  obtains  from  the 
soil. 

The  question  now  arises :  In  what  manner  do  plants  effect  the 
synthesis  of  those  complex  chemical  substances  which  go  to  form 
their  tissues  from  the  simple  bodies  which  serve  as  their  food-stuffs  ? 
The  kinetic  energy  which  is  necessary  to  effect  these  changes  is,  as 
has  been  stated,  derived  from  the  sunlight  and  transformed  into 
potential  energy  by  the  chlorophyl.  We  should  thus  expect  to  find 
in  those  parts  in  which  this  is  present  the  origin  of  those  final 
products  which  we  meet  with  in  the  tissues  of  the  plant.  These 
products  may  be  divided  into  three  groups,  and  in  the  following 
pages  an  attempt  will  be  made  to  describe  the  manner  in  which 
representatives  of  each  are  formed.  I  shall  accordingly  consider 
the  origin  of  the  carbohydrates,  the  fats,  albumins,  and  certain  non- 
albuminous,  nitrogenous  bodies,  all  of  which  are  also  found  in  the 
animal  body,  and  which  represent  the  essential  food-stuffs  of  the 
animal  world.  In  doing  so,  I  am  aware  that  I  am  trespassing  to 
a  certain  extent  upon  what  will  follow  in  subsequent  chapters ;  but 
as  I  shall  deal  with  the  chemistry  of  animal  life  more  exclusively 
in  the  present  work,  it  has  been  deemed  best  to  consider  briefly  the 
principal  syntheses  which  are  effected  by  plants  before  proceeding  to 
a  more  detailed  study  of  the  subject  proper. 

Synthesis  of  the  Carbohydrates, — It  has  been  pointed  out 
that  during  exposure  of  chlorophyl-bearing  plants  to  sunlight 
the  carbon  dioxide  of  the  air  is  decomposed,  with  liberation  of 
oxygen.  The  volume  of  gas  thus  set  free  is  equivalent  to  the 
volume  of  carbon  dioxide  that  is  decomposed.  At  the  same  time  a 
reduction  of  w^ater  takes  place,  as  is  apparent  from  the  observation 


24  INTRODUCTION. 

that  a  larger  amount  of  hydrogen  is  found  in  the  plant  than  is 
necessary  to  form  water  with  all  of  the  oxygen  that  is  present  at  the 
same  time.  It  thus  follows  tliat  one-half  of  the  oxygen  per  volume 
must  be  derived  from  carbon  dioxide,  and  the  otlier  from  water, 
according  to  the  equation  : 

2CO2    +    2H2O    =    2r    -L    40, 
4  volumes.  4  volumes. 

in  which  r  represents  one  atom  of  carbon,  one  atom  of  oxygen,  and 
two  atoms  of  hydrogen,  which  have  been  retained  by  the  plant.  A 
combination  of  these  atoms  in  one  molecule,  however,  would  repre- 
sent one  molecule  of  formic  aldehyde,  CHgO.  Should  this  be 
actually  formed  in  the  plant,  we  would  at  once  have  a  probal)le 
explanation  of  the  manner  in  which  the  carbohydrates  are  con- 
structed, as  these  are  polymeric  compounds  of  formic  aldehyde,  or 
their  anhydrides.  In  fact,  it  is  possible  artificially  to  effect  the 
synthesis  of  many  of  the  carbohydrates  which  are  found  in  the 
living  world  by  starting  "svith  this  simple  aldehyde.  Formic  alde- 
hyde, it  is  true,  has  not  as  yet  been  isolated  as  such  from  the  leaves 
of  jilants,  as  its  existence  here  is  probably  only  momentary,  but  its 
oxidation-product,  formic  acid,  has  frequently  been  obtained.  It  is 
thus  extremely  probable  that  this  is  actually  the  starting-point  in  the 
elaboration  of  the  carbohydrates  by  the  plant.  As  to  the  exact 
manner  in  which  the  aldehyde  is  derived  from  carbon  dioxide  and 
Avater,  we  are  as  yet  uncertain  ;  but  it  appears  from  the  interesting 
researches  of  Gautier  and  Timiriazeff  that  the  chlorophyl  first 
decomposes  water,  under  the  influence  of  sunlight,  and  forms  a 
hydride  of  chlorophyl,  which  is  colorless,  and  that  this  product 
subsequently  reduces  carbon  dioxide,  with  liberation  of  oxygen  and 
restitution  of  the  green  chlorophyl.  These  changes  may  be  rep- 
resented by  the  equations  : 

( 1 )  X  +  H2O  =  xHj  +  O. 

(2)  /H,  +  CO,  =  X  +  H,CO  +  O. 

where  x  represents  the  chlorophyl.  As  a  matter  of  fact,  Gautier 
and  Timiriazeff  succeeded  in  obtaining  such  a  hydride — protophyllin 
— which  turned  green  on  exposure  to  air,  or  in  an  atmosphere  of 
carbon  dioxide  under  the  influence  of  sunlight ;  while  in  the  dark, 
or  on  exposure  to  sunlight  in  an  atmosphere  of  hydrogen,  no  change 
occurred.  The  existence  of  this  body  in  etiolated  plants,  more- 
over, has  likewise  been  established. 

By  a  process  of  polymerization  and  subsequent  hydrolysis,  then, 
formic  aldehyde  gives  rise  to  the  large  number  of  carbohydrates 
which  are  found  in  the  vegetable  world.  Of  the  manner  in  which 
these  polymerizations  and  subsequent  changes  are  brought  about, 
however,  we  know  little ;  but  there  is  reason  to  believe  that  they  are 
largely  effected  through  the  activity  of  special  ferments,  as  has  been 


SYNTHESIS  OF  THE  CARBOHYDRATES.  25 

indicated.  That  some  of  these  changes  take  place  in  tlie  cliloropliyl- 
bearing  parts  of  the  plant  can  readily  be  demonstrated.  If  a  spiro- 
gyra,  for  example,  is  exposed  to  snnlight  after  having  been  kept  in 
darkness  for  some  time,  so  as  to  remove  any  sugar  that  may  have 
been  present  in  the  cell,  it  will  be  observed  that  after  a  very  few- 
minutes  starch  granules  appear,  which  can  readily  be  detected  by  the 
addition  of  a  little  iodine  solution.  On  subsequent  removal  from 
the  light  the  starch  soon  disappears  from  the  green  parts  of  the 
plant,  and  is  carried  to  the  storage-cells  proper,  where  it  is  trans- 
formed into  dextrin,  glucose,  and  various  soluble  gums,  which  may 
be  further  transformed  into  celluloses,  certain  mucilages,  etc. 

Glucosides. — Closely  related  to  the  carbohydrates  proper,  the 
origin  of  which  has  just  been  considered,  is  a  group  of  substances 
which  likewise  occur  widely  distributed  in  the  vegetable  kingdom. 
These  are  the  so-called  glucosides.  They  are  so  termed  from  the 
fact  that  glucose  is  invariably  formed  during  their  hydrolytic 
decomposition,  which,  as  an  anhydride,  thus  constitutes  an  integral 
part  of  their  molecule.  This  observation  at  once  suggests  their 
origin  also  from  formic  aldehyde. 

Such  substances  are  salicin,  which  on  hydrolytic  decomposition 
yields  glucose  and  saligenin  ;  arbutin,  which  yields  glucose  and 
hydroquinon  ;  phloridzin,  which  gives  rise  to  glucose  and  phlore- 
tin,  etc. 

(1)  C.jHisOr     +   H,0  =   C^HgO^      +   C«H,A- 

Salicin.  Saligenin.  Glucose. 

(2)  C„H,eOj     +    H,0   =^   CgHeO,      +   CeK^.p,. 
Arbutin.  Hydroquinon.       Glucose. 

(3)  a.H^p.o   +    11,0   =   C,,H,A   +    CeH.A- 
Phloridzin.  Phloretin.  Glucose. 

Especially  interesting  is  a  group  of  glucosides  which  are  nitro- 
genous in  character,  and  thus  stand,  as  it  were,  midway  between  the 
carbohydrates  and  the  albumins.  A  study  of  their  decomposition- 
products  hence  permits  an  insight  into  the  manner  in  which  the 
albumins  are  synthetically  produced,  and  shows  that  here  also  alde- 
hyde groups  play  an  important  part.  As  in  the  case  of  the  albu- 
mins, the  nitrogen  here  also  occurs  in  combination  with  carbon  and 
hydrogen  in  the  group  CH — NH,  which  in  turn  is  structurally  closely 
related  to  hydrocyanic  acid.  In  accordance  with  these  considera- 
tions, we  thus  find  that  amygdalin,  C^oHg^NOn,  is  decomp^sed  into 
glucose,  hydrocyanic  acid,  and  benzaldehyde.  Solanin,  (^43N.jjNOie, 
similarly  yields  glucose  and  solanidin,  C25H39NO. 

Mannides. — Like  the  carbohydrates  proper,  the  mannides  or  nian- 
nitides,  which  also  occur  widely  distributed  in  the  vegetable  world, 
are  likewise  derived  from  the  aldehyde  radicle  that  is  formed  by 
chlorophyl  under  the  influence  of  sunlight.  They  differ  frt)m  the 
gluco.sides  in  yielding  maniiite,  CgH,^Og,  instead  of  glucose,  on 
hydrolytic  decomposition.  The  origin  of  mannite  from  formic  alde- 
hyde may  be  represented  by  the  equation  : 


(1) 

3C0j 

(2) 

34CO2 

(3) 

C3H5(OH). 
Glvcerin. 

26  ISTRODUCTIOX. 

eCHjO  -r  111  =  C'eHuOe. 

On  the  other  hand,  mannite  may  result  from  glucose  as  the  result 
of  the  specific  activity  of  certain  cells,  as  is  shown  by  the  equation  : 

ISCgHi.bs  -  OH.O  =  12C6H,A  -j-  GCO^. 

Synthesis  of  the  Fats. — The  fats  which  are  found  in  plants 
are,  like  the  carljohydrates,  derived  from  carl)on  dioxide  and  water, 
and  in  all  likelihood  are  formed  also  synthetically  through  the 
agency  of  the  chlorophyl.  The  mechanism,  however,  by  which 
these  syntheses  are  effected  is  not  so  clear.  It  is  probable  that  they 
result  from  the  union  of  carbon  dioxide  and  water,  as  shown  by  the 
equations  : 

4H2O  =  C3H  A         ■  -r  70. 

Glycerin. 

34H,0  =  CigHg^O,  +  I6CH2O,  —  680. 

Stearic  acid.  Formic  acid. 

X,8H350.0H  =  C3H5(O.C,gH350)3  ^  SHjO. 

Stearic  acid.  Stearin. 

This  supposition  is  strengthened  by  the  observation  that  during 
certain  plia.ses  in  the  life  of  some  plants  an  actual  transformation 
of  carbohydrates  into  fats  takes  place.  In  the  fruits  and  leaves  of 
the  olive  tree,  for  example,  a  large  amount  of  mannite  gradually 
disappears  during  the  months  of  September  and  October,  and  is 
replaced  by  oil.  This  transformation  could  be  explained  upon  the 
basis  of  the  equations  just  given,  or  by  the  assumption  that  the  oil 
results  from  mannite  through  a  loss  of  water  and  carbon  dioxide,  as 
suggested  by  the  equation  : 

llCgHiA  -  Q.H^Os  -  30H,O  -  loCOj. 

In  any  event,  the  system  HoO  —  CO,,  which  gives  rise  to  the 
formatirin  of  formic  aldehyde  and  glucose,  must  also  be  regarded 
as  the  fundamental  basis  in  the  synthesis  of  fats. 

Synthesis  of  the  Albumins. — Much  more  complicated  than  the 
synthesis  of  the  carbohydrates  and  fats  is  that  of  the  albumins, 
a  class  of  bodies  which  occur  widely  distributed  in  both  the  animal 
and  the  vegetal)le  world,  and  form  the  groundwork,  so  to  speak,  of 
all  living  matter.  Like  the  carljohydrates  and  fats,  they  also  con- 
sist of  cgrbon,  hydrogen,  and  oxygen,  but  in  addition  to  these  ele- 
ments nitrogen  and  variable  amounts  of  sulphur  are  constantly 
present.  To  this  cla.ss  belong  such  bodies  as  .serum-albumin,  egg- 
albumin,  casein,  fibrin,  etc.  They  are  exceedingly  complex  sub- 
stances, and  have  a  very  high  molecular  weight. 

The  exact  manner  in  which  the  albumins  originate  has  not  been 
determined,  and  the  many  attempts  wiiich  have  been  made  to  effect 
the  synthesis  of  bodies  belonging  to  this  class  have  not  led  to  any 
definite  results.  We  are  in  possession  of  a  number  of  observations, 
however,  which  permit  an  insight,  at  least,  into  the  manner  in 
which  plants  are  capable  of  elaborating  these  complex  substances 


SYNTHESIS  OF  THE  ALBUMINS.  27 

from  the  simple  material  which  serves  as  their  food,  and  there  is 
reason  to  suppose  that  the  synthesis  of  the  albumins  also  takes 
place,  to  a  certain  extent  at  least,  in  the  chlorophyl-bearing  por- 
tions of  plants. 

It  was  formerly  supposed  that  the  nitrogen  necessary  in  these 
synthetic  processes  was  furnished  plants  in  the  form  of  ammonium 
salts.  Subsequent  investigations  have  shown,  however,  as  has  been 
indicated,  that  this  is  usually  not  the  case,  and  we  now  know  that 
through  the  activity  of  various  bacteria  in  the  soil  the  nitrogen 
required  by  plants  is  here  oxidized  to  nitrates.  These  are  absorbed 
and  carried  to  the  chlorophyl-bearing  portions  of  the  plant,  where, 
as  we  have  seen,  formic  aldehyde  and  glucose  are  constantly  being 
formed.  Here,  or  in  the  rootlets,  a  certain  proportion  of  the  nitrates 
is  apparently  transformed  into  nitric  acid,  which  is  then  promptly 
reduced  by  the  formic  aldehyde,  with  the  formation  of  a  certain 
amount  of  hydrocyanic  acid,  as  shown  in  the  equation  : 

2HNO3  +  5CH2  O  =  2HCN  +  3C0,  +  5H2O. 

In  this  form,  then,  the  nitrogen  probably  enters  into  the  construc- 
tion of  the  albuminous  molecule.  This  supposition  is  strengthened 
by  the  observation  that  hydrocyanic  acid,  as  such,  or  in  the  form  of 
cyanides,  occurs  widely  distributed  in  the  vegetable  world,  and  is 
characterized  by  the  readiness  with  which  it  coml)ines  with  a  large 
number  of  organic  substances  to  form  highly  comj^lex  chemical  com- 
pounds. The  nature  of  the  subsequent  changes  will  be  better  under- 
stood when  the  decomposition-products  of  the  albumins  have  been 
studied  in  detail.  These  will  be  considered  in  a  section  of  the 
following  chapter.  Qualitatively  they  are  the  same  in  plants  and 
animals,  and  there  is  reason  to  assume  that  the  structure  of  the 
albumins  also  is  analogous  in  both  classes  of  living  matter. 


CHAPTER    II. 

THE  ALBUMINS 

The  albumins,  or  proteins,  are  the  most  important  food-stuffs 
which  the  animal  requires  for  its  existence.  They  play  a  pre- 
dominating role  in  the  construction  of  all  the  tissues  and  organs  of 
the  body,  and  form  the  groundwork  of  every  living  cell.  The 
phenomena  of  life  depend  upon  and  centre  in  their  presence. 

While  many  different  forms  of  albumins  exist,  they  all  present 
certain  general  chemical  and  physical  characteristics,  Mhich  serve 
to  distinguish  them  as  a  class,  and  which  show  that  a  close  genetic 
relationship  exists  between  them. 

Elementary  Composition. — All  albumins  contain  carbon,  hydro- 
gen, nitrogen,  oxygen,  and  sulphur  in  certain  definite  proportions, 
which  vary  but  little  in  the  different  members  of  the  group.  The 
variations  which  do  occur  are  shown  in  the  following  table : 

Carbon 50.0-55.0  per  cent. 

Hydrogen 6.5-  7.3    "      '' 

Nitrogen .  15.0-17.6    "      " 

Oxygen 19.0-24.0    "      " 

Sulphur 0.3-  2.4    "      " 

Other  elements  are  not  found  in  the  common  albumins,  but  occur 
in  certain  compound  albumins,  which  result  through  the  union  of 
an  albuminous  group  with  other  more  or  less  complex  radicles. 
The  haemoglobins  thus  contain  iron,  the  nucleoproteids  phosphorus  ; 
a  certain  highly  differentiated  globulin  contains  iodine,  etc.  All 
albumins  further  contain  variable  amounts  of  mineral  salts,  Mhich 
are  closely  united  with  the  albuminous  molecule.  The  most  im- 
portant and  constant  of  these  are  the  chlorides  and  phosphates  of 
the  alkalies  and  the  alkaline  earths. 

The  structural  formula  of  the  albumins  is  unknown.  The  mole- 
cule is  unquestionably  very  large,  hoAvever.  In  the  case  of 
serum-albumin  Hofmeister  calculated  the  general  formula  as 
C^-i)H72oNj,gS,;0,^()5  "which  would  correspond  to  a  molecular  weight 
of  10,166.  (See  molecular  weight  and  structural  composition,  below.) 

Reaction. — In  the  free  state  the  albumins  are  neutral ;  in  such 
solutions  they  are  not  ionized,  but  this  results  at  once  on  contact 
with  other  ions.  They  then  form  salts,  both  with  acids  and  bases 
which  are  fairly  good  conductors,  and  thus  ]>lay  the  part  of  pseudo- 
acids  and  pseudo-bases  (Hantzsch).  As  acids  the  albumins  are 
dibasic  (Osborne,  Soldner,  and  others).  One  series  of  salts  shows  a 
neutral  reaction  with  the  usual  indicators,  while  the  other  is  more 

28 


CRYSTALLIZATION.  ^9 

or  less  markedly  basic.  The  acid  character  is  most  pronounced  in 
the  mucins,  the  nucleo-albumins,  and  the  nucleoproteids  ;  less  so  in 
the  globulins ;  while  the  albumins  proper  are  apparently  neutral 
or  slightly  alkaline.  The  histons  and  the  protamins,  on  the  other 
hand,  are  markedly  basic. 

Solubility. — Some  albumins  are  soluble  in  water,  others  only  in 
dilute  saline  solution,  and  still  others  in  dilute  acids  and  alkalies. 
In  more  concentrated  acids  and  alkalies,  as  also  in  glacial  acetic 
acid,  all  albumins  dissolve,  but  are  at  the  same  time  decomposed. 
In  dilute  alcohol  some  albumins  dissolve  with  comparative  ease, 
but  in  absolute  alcohol,  ether,  chloroform,  benzol,  and  all  other 
common  solvents  they  are  insoluble. 

Crystallization. — In  the  eggs  of  certain  fishes  and  amphibia  so- 
called  i/olk  platelets  may  be  observed  which  apparently  possess  a 
crystalline  structure.  Chemical  examination  has  shown,  however, 
that  these  bodies  are  not  pure  albumins,  but  that  they  contain  a 
large  percentage  of  lecithin  and  mineral  salts.  The  same  is  true 
of  the  alenronat  crystals  which  have  been  found  in  the  seeds  of  cer- 
tain plants,  and  probably  also  of  the  eosinophilic  cri/stalloids  which 
may  be  seen  in  the  blood  of  birds. 

The  form  in  which  the  albumins  are  commonly  obtained  in  the 
chemical  laboratory,  and  in  which  (with  the  exceptions  mentioned) 
they  exist  in  nature,  is  amorphous.  In  the  dry  state  they  form  a 
white  or  but  little  colored  non-hygroscopic  powder,  or  they  occur 
as  yellowish,  brittle,  more  or  less  opaque  lamellse  which  are  both 
odorless  and  tasteless. 

By  artificial  means  it  is  possible  to  obtain  certain  members  of 
the  group  in  crystals.  To  this  end,  a  neutral  salt  must  be  present, 
and  it  is  advantageous  to  have  the  reaction  slightly  acid.  What 
part  the  salt  and  the  acid  play  is  not  known,  but  there  is  evidence 
to  show  that  either  the  acid  or  the  salt  enters  into  the  composition 
of  the  crystals.  In  the  case  of  edestin  Osborne  has  shown  that  a 
salt-like  body  results,  which  contains  hydrochloric  acid  when  crys- 
tallization has  taken  place  from  a  solution  of  the  albumin  in  sodium 
chloride,  or  sulphuric  acid  if  a  sul])hate  has  been  employed. 

Of  animal  albumins,  crystallization  has  been  notably  eifected  in 
serum-albumin,  egg-albumin,  and  lactalbumin.  Artificial  crystal- 
lization of  globulins  has  not  led  to  satisfactory  results ;  but  one  sub- 
stance which  supposedly  belongs  to  this  class,  and  which  was  noted 
by  Noel  Paton  in  a  pathological  urine,  separated  out  spontaneously 
in  crystalline  form. 

Of  vegetable  albumins,  the  edestin  of  hempseed,  the  phytovitellin 
of  Paranuts,  and  the  castor-oil  bean  are  notable  examples  of  crystal- 
lizable  albumins. 

The  form  of  the  crystals  seems  to  vary  ;  but  Wichmann  has 
shown  that  they  are  all  crvstallographically  identical,  or  at  least 
isomorphous.  They  probably  belong  to  the  hexagonal  system  and 
are  more  or    less    markedly  doubly   refractive   (positively).     The 


30  THE  ALBUMINS. 

most  perfect  forms  are  obtained  on  repeated  crystallization,  ^vliile 
as  intermediary  forms  spheroids  and  globnlits  are  commonly  encoun- 
tered. If  the  crystallization  is  repeated  too  often,  the  crystalline 
appearance  niav  be  lost  and  the  body  again  becomes  amorphous. 

Diffusion. — Like  the  colloids  of  the  inorganic  world,  so  also  are 
the  albumins  incapable  of  ditfusing  through  animal  membranes  or 
vegetable  parchment.  This  peculiarity  Graham  explained  by  the 
assumption  that  such  bodies  do  not  occur  in  a  state  of  actual  solution. 
This,  however,  is  not  the  case,  for  it  has  been  shown  that  albu- 
minous solutions  are  capable  of  conducting  the  electrical  current 
and  may  exist  both  as  anions  and  kations — /.  c,  that  they  are  true 
solutions.  Their  inability  to  pass  through  animal  membranes  is 
explained. most  likely  by  the  size  of  the  allMuniuous  molecule.  This 
property  is  very  important  from  the  standpoint  of  chemical  tech- 
nique, as  it  renders  it  possible  to  separate  the  albumins  from  a 
large  number  of  other  bodies  which  may  simultaneously  be  present 
in  solution. 

Behavior  toward  Polarized  Light. — All  true  albumins  are 
Isevorotatory,  the  degree  of  rotation  being  different  in  different 
members  of  the  group.  This  fact  has  been  utilized  in  the  identi- 
fication of  the  individual  albumins,  but  it  is  to  be  noted  that  unless 
the  examinations  can  be  made  with  neutral  aqueous  solutions  the 
resulting  data  will  not  be  constant.  Biilow  has  definitely  proved 
that  the  same  albuminous  solution  will  show  a  varying  degree  of 
rotation  with  a  varying  reaction.  Some  of  the  results  which  have 
been  obtained  are  given  in  the  following  table  : 

Animal  albumins.  Vegetable  albumins  (Osborne). 

8erum-albumin  (a)D  ....  — 56°  Edestin         (hempseedj 

Serum-globulin — 59°-75°  (a)D —41.3° 

Fribrinogen —43°  Globulins            (various 

Egg-albumin — 33°-38°  forms) — 38.78°-45.21° 

Lactalbumin — 36°-37°  Excelsin  (Brazilnut)     .  —42.94° 

Casein  (in  MgSO^  solution)    .  — 80°  Amandin  (almonds)  .    .  — 56.44° 

Syntonin  (from  myosin)      .    .  —72°  Corylin  (filbert)      .    •    •  —43.09° 

Alkaline  albuminate    .    .    .    .—02.2°  Zem  (maize) —28.00° 

Various  albumoses — 70°-80°  Gliadin  (wheat)     .    .    .  —92.28° 

Phaseolin  (kidney  bean)  — 41.46° 

While  Igevorotation  is  constant  in  the  true  albumins,  dextrorota- 
tion probably  occurs  in  all  nucleoproteids.  This  has  been  estab- 
lished directly  in  the  case  of  the  nucleoproteids  of  the  pancreas, 
the  thymus  aiid  tlie  adrenal  glands  (Gamgee,  Jones),  and  Osborne 
has  sliown  that  tliis  property  is  very  likely  AvhoUy  referable  to 
the  nucleinic  acid  component.'  The  degree  of  dextrorotation  in  the 
substances  which  thus  far  have  been  examined  varied  between 
-f37.5°  (nuclcohiston  from  thymus)  and  97.9°  (Hammarsten's 
/9-nucleopr()t('id  of  the  pancreas). 

Coagulation. — One  of  the  most  characteristic  properties  of  tlie 
albumins  is  the  pliysical  instability  of  their  solutions  and  their 
mark(;d  tendency  to  revert  to  a  solid  or  semisolid  state.     This 


COAGULATION.  31 

enables  them  to  play  the  important  part  which  they  take  in  the 
construction  of  the  various  tissues,  and  no  doubt  renders  possible 
the  manifold  and  chemically  often  antagonistic  reactions  which 
may  simultaneously  occur  within  the  bodies  of  the  individual  cells. 
This  change  may  be  effected  by  apparently  trivial  factors,  such  as 
evaporation,  contact  with  porous  substances,  etc.  In  this  respect 
also  the  albumins  behave  very  much  like  the  inorganic  colloids. 
When  a  solution  of  sodium  silicate  is  thus  added  to  large  excess  of 
dilute  hydrochloric  acid,  the  silicic  acid  which  is  formed  is  apj)ar- 
ently  held  in  solution.  If  then  the  excess  of  hydrochloric  acid, 
together  with  the  sodium  chloride  formed  during  the  reaction,  is 
removed  by  dialysis,  a  clear  solution  of  silicic  acid  remains 
in  the  dialyzer.  This  is  transformed  at  once  into  a  thick, 
gelatinous  material  when  a  small  amount  of  carbon  dioxide  is 
passed  through  the  solution.  Some  of  the  albumins,  such  as  the 
globulins,  behave  in  a  similar  way.  In  undergoing  such  changes 
the  albumins  may  retain  their  original  properties  for  a  while  at 
least,  but  after  a  variable  period  they  become  insoluble,  and  are 
then  said  to  be  coagulated.  This  change  can  be  brought  about  at 
once  by  the  application  of  heat,  and  it  is  important  to  note  that 
all  true  native  albumins  can  be  coagulated  in  this  manner.  After 
coagulation  their  solution  can  only  be  eifected  by  influences  which 
lead  to  their  more  or  less  extensive  destruction.  They  have  lost 
those  physical  properties  which  characterized  them  individually  as 
albumins ;  they  are  permanently  denaturized,  as  Neumeister  ex- 
presses it. 

The  temperature  of  coagulation  differs  with  the  different  albu- 
mins, and  is  fairly  constant  for  the  individual  bodies,  providing 
that  the  reaction  of  the  solution  is  neutral,  or  still  better  very 
faintly  acid.  If  the  reaction  is  alkaline,  coagulation  is  not  com- 
])lete,  and  in  the  presence  of  more  than  traces  of  free  alkali  or  an 
alkaline  carbonate  it  may  not  occur  at  all.  A  markedly  acid  reac- 
tion interferes  in  a  like  manner.  Equally  important  is  tlie  presence 
of  a  certain  amount  of  salt.  Cohnheim  explains  these  peculiarities 
as  follows :  On  heating  the  albuminous  solution  the  albumin  is 
denaturized,  no  matter  what  the  reaction  may  be  or  whether  salts 
are  present  or  absent.  In  the  presence  of  alkalies  the  denaturized 
albumins  form  alkaline  albuminates,  which  are  readily  soluble  in  the 
case  of  the  alkalies  and  with  diflficidty  so  in  the  case  of  the  alkaline 
earths.  With  an  acid  reaction,  on  the  other  hand,  acid  albumin  is 
formed,  viz.,  the  hydrochlorate,  or  acetate,  of  the  denaturized  albu- 
mins as  the  case  may  bo ;  this  by  itself  is  soluble  in  water,  but 
insoluble  in  the  presence  of  salts. 

Spiro  has  recently  demonstrated  that  the  temperature  of  coagula- 
tion is  materially  influenced  by  the  presence  of  certain  nitrogenous 
bodies,  such  as  cholin,  piperidin,  pyridin,  and  anilin,  which  are  all 
capable  of  preventing  the  coagulation  of  a  certain  amount  of  albumin. 
Urea  is  even  more  active  in  this  respect,  and  when  present  in  suffi- 


32  THE  ALBUMINS. 

cient  concentration  may  prevent  coagulation  altogether.  A  similar 
effect  ift  protluced  by  the  mustard  oils.  The  reason  of  this  is  not 
clear,  nor  is  it  possible  to  offer  a  satisfactory  explanation  of  the  phe- 
nomenon of  coagulation  in  general. 

Formerly  the  separation  of  the  different  albumins  from  each 
other  and  their  identification  by  fractional  coagulation  was  ex- 
tensively employed,  while  at  present  the  method  has  fallen  some- 
what into  disfavor.  This  is  largely  owing  to  the  observation  that 
the  temperature  of  coagulation  may  vary  Mithin  fairly  wide  limits 
with  the  amount  of  salt  present  and  the  reaction.  It  is  to  be  noted, 
however,  that  with  constant  conditions  as  regards  these  two  factors, 
and  especially  in  reference  to  the  reaction,  the  result  also  will  l)e 
quite  constant.  To  insure  complete  precipitation,  the  reaction 
should  be  just  acid  ;  if  this  point  has  been  attended  to,  the  amount 
of  salt  is  of  secondary  importance,  providing  that  not  too  little  is 
present.  In  any  event,  in  reporting  results  it  is  well  to  note  the 
concentration  of  the  albuminous  solution,  the  strength  of  the  salt 
solution,  and  the  reaction,  together  with  the  temperature  of  coagu- 
lation. 

The  coagulation-]3oints  of  the  different  albumins  are  mentioned 
under  their  respective  headings. 

Denaturization. — It  has  been  pointed  out  that  heat  coagulation 
alters  the  character  of  all  true  albumins  in  such  manner  that  they 
lose  their  common  properties  and  are  wo  longer  soluljle  in  the  usual 
neutral  media.  Xeumeister  has  termed  the  change  denaturization 
of  the  albumins.  This  change  can  also  be  brought  about  by  other 
means  than  heat,  such  as  precipitation  with  acids,  the  salts  of  the 
heavy  metals,  the  various  alkaloidal  reagents,  shaking  with  chloro- 
form or  ether,  and  even  by  prolonged  standing.  Coagulation  is  not 
an  essential  phase  of  denaturization.  Denaturization  may  indeed 
become  manifest  by  the  non-occurrence  of  coagulation.  This  occurs 
upon  the  addition  of  metallic  silver  or  formalin  to  all>uniinous 
solutions,  when  coagulation  is  no  longer  possible.  The  albumins 
in  question  are  then  only  held  in  solution  if  the  reaction  is  acid  or 
alkaline  ;  in  a  neutral  solution  they  are  precipitated.  Denaturized 
and  coagulated  albumins  can  only  be  brought  into  solution  by 
means  which  will  at  the  same  time  produce  integral  changes  in 
their  composition,  viz.,  by  means  of  proteolytic  ferments,  dilute 
mineral  acids  or  alkalies,  concentrated  organic  acids  under  the 
application  of  heat,  etc.  Acid  albumins  (syntonins)  and  alkaline 
albuminates  result  which  on  further  exposure  to  ferments,  mineral 
acids,  or  alkalies  are  transformed  into  alburaoses,  and  these  in  turn 
into  less  complex  bodies,  which  are  collectively  spoken  of  as 
peptones. 

The  nature  of  the  ])rocess  which  determines  denaturization  is 
possibly  a  primary  cleavage.  In  favor  of  such  a  view  is  the 
observation  that  coagulated  albumin  is  mr)re  readily  hydrolized  by 
ferments.     Nothing  certain,  however,  is  known. 


BEHAVIOR   TOWARD  NEUTRAL  SALTS.  33 

Behavior  toward  Neutral  Salts. — All  albumins  and  albumoses 
can  be  precipitated  from  their  solutions  by  means  of  certain  neutral 
salts,  without  loss  of  their  characteristic  properties  or  change  in 
structure.  The  resulting  precipitates  are  soluble  as  before,  Tliis 
behavior  toward  neutral  salts  is  not  characteristic  of  albumins, 
however,  as  every  substance  can,  generally  speaking,  be  withdrawn 
from  its  solution  by  a  second  body,  but  owing  to  the  great  molec- 
ular size  of  the  albuminous  molecule  these  bodies  are  thrown  out 
of  solution  more  readily  than  others. 

The  salts  which  are  usually  employed  in  the  chemical  laboratory 
for  the  precipitation  of  the  albumins  are  sodium  chloride,  sodium 
sulphate,  magnesium  sulphate,  and  especially  ammonium  sulphate 
and  zinc  sulphate.  The  two  latter  are  especially  important,  as  they 
are  universal  precipitants,  while  the  other  salts  and  many  others, 
which  have  not  been  mentioned,  will  throw  down  only  certain  in- 
dividual albumins.  Generally  speaking,  the  more  highly  differen- 
tiated albumins  can  be  precipitated  by  the  least  active  salts,  such  as 
sodium  chloride  and  magnesium  sulphate,  while  for  the  complete 
precipitation  of  the  less  complex  albumins  (serum-albumin)  and 
certain  albumoses  ammonium  sulphate  or  zinc  sulphate  is  necessary. 
The  more  complex  the  albumin  the  more  readily  it  is  precipitated, 
so  that  with  certain  bodies,  such  as  fibrinogen,  casein,  and  other 
nucleo-albumins  complete  saturation  is  not  necessary.  In  their 
behavior  toward  ammonium  sulphate  these  bodies  differ  from  the 
less  complex  albumins  in  the  fact  that  they  require  a  smaller  amount 
of  the  salt  for  their  precipitation.  The  simpler  the  structure,  on 
the  other  hand,  the  more  salt  is  necessary.  The  deutero-albumoses, 
which  probably  represent  the  least  complex  bodies,  which  still  have 
a  distinct  albuminous  character,  can  only  be  precipitated  by  com- 
plete saturation  with  ammonium  or  zinc  sulphate,  and  with  one  of 
their  number  it  is  necessary  to  carry  out  the  saturation  in  acid 
solution,  while  all  other  albumins  and  albumoses  can  be  thrown 
down  from  their  neutral  solutions. 

Magnesium  sulphate  occupies  a  position  intermediate  between 
sodium  chloride  and  ammonium  sulphate,  and  readily  precipitates 
both  globulins  and  primary  albumoses. 

The  behavior  of  the  different  albumins  to  neutral  salts,  and 
notably  to  ammonium  and  zinc  sulphate,  is  now  largely  utilized  in 
their  differentiation  from  each  other  and  the  identification  of  the  in- 
dividual bodies.  It  has  been  ascertained  that  under  certain  con- 
ditions each  albumin  has  a  definite  lower  and  upper  limit  of  precip- 
itation, which  for  that  body  is  quite  constant — /.  e.,  for  a  given 
volume  of  the  solution  a  definite  amount  of  salt  is  necessary, 
beneath  which  no  precipitation  occurs — the  lower  limit,  and  beyond 
which  no  further  precipitation  takes  place — the  upper  limit.  In 
conformity  with  Hofmeister's  suggestion  this  examination  is  now 
generally  conducted  as  follows :  A  series  of  test-tubes  is  prepared 
which  are  successively  charged  with  a  constant  number  of  c.c.  (2) 
3 


34  THE  ALBUMINS. 

of  the  albuminous  solution  (2  per  cent.)  and  increasing  amounts  of 
a  saturated  neutral  solution  of  ammonium  sulphate,  the  mixture 
being  diluted  in  each  case  to  10  c.c.  Avith  distilled  water.  The  con- 
tents of  each  tube  are  thoroughly  mixed  and  set  aside  for  one-half 
hour,  when  a  further  examination  is  made.  In  the  first  series  it  is 
well  to  start  with  a  set  of  tubes  containing  0.5,  1.0,  1.5,  2.0,  2.5, 
etc.,  c.c.  of  the  sulphate  solution.  At  the  end  of  a  half  hour  the 
contents  of  each  tube  are  passed  through  small  filters  and  the 
filtrate  treated  with  an  additional  ^  c.c.  of  the  salt  solution.  Sup- 
posing that  tube  1  containing  0.5  c.c.  presented  no  turbidity,  but 
that  this  was  marked  in  tube  2,  then  it  is  manifest  that  the  lower 
limit  of  precipitation  lies  between  0.5  and  1.  If  now  the  fihrate 
of  tube  1  after  the  addition  of  0.1  c.c.  of  the  salt  solution  and 
standing  shows  a  turbidity,  the  lower  limit  for  this  particular  albumin 
is  called  0.6.  In  a  similar  manner  the  upper  limit  is  determined. 
Supposing  that  the  filtrate  of  the  tube  containing  3.5  c.c.  of  salt 
solution  becomes  turbid  on  adding  0.1  c.c.  further,  while  in  the 
filtrate  of  a  tube  containing  3.6  c.c.  salt  solution  from  the  first,  no 
turbidity  developed,  it  is  clear  that  3.6  represents  the  upper  limit. 

In  the  following  table  the  limits  of  precipitation  of  some  of  the 
more  important  albumins  have  been  collected  : 

Lower  limit.  Upper  limit. 

Nucleoalbumins             0.1-0.6  1.6-2.3 

Fibrinogen 1.5-1.7  2.5-2.7 

Fibrinoglobulin 2.2  2.9 

Euglobnlin 2.8  3.3 

Rseudoglobulin 3.4  4.6 

Seruni-albumin 6.4  9.0 

Especially  characteristic  and  constant  is  the  upper  limit,  while 
the  lower  limit  occasionally  fluctuates  somewhat.  In  acid  solu- 
tions there  is  a  general  lowering  of  the  limits  of  precipitation  ;  in 
this  case  the  albumins  are  not  precipitated  as  such,  but  as  salts,  the 
albumins  playing  the  part  of  a  base. 

After  precipitation  the  albumins  tenaciously  hold  a  certain 
amount  of  the  salt,  which  can  scarcely  be  removed,  even  on  \)V0- 
longed  dialysis.  Noteworthy  also  is  the  fact  that  certain  albumins 
are  capable  of  abstracting  the  acid  of  the  particular  salt  which  is 
employed,  to  form  compounds  from  which  the  acid  in  question 
cannot  be  removed  on  washing  with  water.  This  peculiarity  is 
especially  well  marked  in  the  case  of  serum-albumin,  and  ])robably 
accounts  for  the  observation  that  on  repeated  crystallization  from 
ammonium  sulphate  solution  the  mixture  of  albumin  and  sulphate 
becomes  more  and  more  acid,  while  at  the  same  time  ammonia  is 
liberated  (G.  Meyer).  Such  acid  compounds  of  proteins  can  appar- 
ently also  occur  in  nature,  as  has  been  shown  in  the  case  of  horn 
and  human  hair  (Morner).  This  property  of  abstracting  acids  from 
the  corresponding  salts  and  uniting  therewith  should  not  be  con- 
founded with  the  power  of  certain  albumins  of  combining  with 
free  acids  directly,  which  has  been  demonstrated  by  Sjoqvist,  Cohn- 


SPECIAL  REACTIONS  OF  THE  ALBUMINS.  35 

heim,  and  others.     These  compounds  orighiate  only  in  the  presence 
of  free  acids  as  such. 

Behavior  toward  Alcohol. — While  some  of  the  albumins 
(albumoses)  dissolve  in  dilute  alcohol  with  comparative  ease,  strong 
alcohol  acts  in  much  the  same  manner  as  the  neutral  salts.  But 
it  is  to  be  noted  that  after  prolonged  exposure,  and  especially  in 
the  presence  of  salts,  the  albumins  are  coagulated,  and  then  remain 
refractory  to  all  neutral  solvents. 

Special  Reactions  of  the  Albumins. 

Precipitation. — It  has  been  pointed  out  that  with  the  exception 
of  the  peptones  practically  all  albumins  can  be  precipitated  from 
their  neutral  or  feebly  acid  solutions  by  certain  neutral  salts.  As 
a  result  of  this  process  they  apparently  undergo  no  change  in 
structure  or  in  their  general  properties,  and  remain  soluble  in  the 
usual  neutral  media.  There  is  a  large  number  of  substances,  how- 
ever, which  also  precipitate  the  albumins,  but  which  either  cause 
their  coagulation  or  combine  with  them  to  form  compounds  which 
are  insoluble  in  water.  Many  of  these  reagents  are  extensively  used 
in  the  chemical  laboratory  in  testing  for  albumins  ;  they  furnish  reac- 
tions which  individually  are  not  absolutely  characteristic,  but  the 
albuminous  nature  of  a  substance  can  usually  be  regarded  as  estab- 
lished when  a  positive  result  is  obtained  with  all  or  at  least  the 
larger  number  of  the  reagents.  The  reactions  are  common  to  the 
true  albumins,  the  proteids,  and  the  majority  of  the  albumoses. 

The  most  important  reagents  are  the  following : 

1.  The  mineral  acids,  viz.,  nitric,  hydrochloric,  sulphuric,  and 
metaphosphoric  acid.  These  are  employed  in  concentrated  form. 
The  one  most  commonly  in  use  is  nitric  acid.  The  testis  conducted 
by  allowing  a  small  amount  of  the  acid  to  flow  beneath  the  fluid  to 
be  tested,  when  a  white  ring  of  coagulated  albumin  appears  at  the 
zone  of  contact  (Heller's  test).  In  the  case  of  the  true  albumins, 
all  of  which  give  the  reaction,  the  precipitate  is  insoluble  in  an 
excess  of  the  acid,  even  on  heating.  Bat  with  the  albumoses,  which 
in  part  are  also  precipitated,  the  precipitate  dissolves  on  boiling 
and  reappears  on  cooling. 

2.  The  Salts  of  the  Heavy  Metals. — In  combining  with  these  the 
albumins  play  the  part  of  weak  organic  acids.  They  set  free  the 
corresponding  acids  of  the  salts  and  combine  with  the  metallic 
oxides  to  form  compounds  which  are  insoluble  in  neutral,  alkaline, 
and  acid  solutions.  With  the  exception  of  myogen,  hemoglobin, 
and  certain  albumoses,  the  resulting  precipitates  are  as  a  rule  insolu- 
ble in  an  excess  of  the  reagent. 

The  salts  which  are  usually  employed  are  the  sulphate  and  ace- 
tate of  copper,  the  chloride  and  acetate  of  iron,  the  neutral  and 
basic  acetate  of  lead,  the  bichloride  of  mercury,  nitrate  of  silver, 
acetate  of  uranium,  acetate  of  zinc,  chloride  of  platinum,  etc.     All 


36  THE  ALBUMINS. 

of  these  may  be  used  in  moderately  concentrated  solution,  and  are 
added  directly  to  the  albuminous  fluid. 

Especially  important  are  the  salts  of  iron,  copper,  and  lead.  If 
ferric  chloride  is  added  to  an  albuminous  solution  containing  an 
excess  of  sodium  acetate,  until  a  distinct  red  color  results,  the  albu- 
mins are  completely  precipitated  on  boiling.  An  excess  of  the  iron 
must  be  avoided,  as  the  precipitated  albumins  will  otherwise  dis- 
solve. 

Acetate  of  copper  precipitates  all  true  albumins  and  serves  to 
separate  the  primary  from  the  secondary  albumoses  ;  an  excess 
should  be  avoided. 

On  boiling  albuminous  solutions  with  hydroxide  of  lead  in  the 
presence  of  acetate  of  lead  complete  precipitation  occurs. 

Bichloride  of  mercury  precipitates  not  only  the  true  albumins 
and  albumoses,  but  also  the  peptones. 

The  Alkaloidal  Reagents. — The  most  important  of  these  are  phos- 
photungstic  and  phosphomolybdenic  acid,  mercuropotassic  iodide, 
bismuthopotassic  iodide,  and  cadmium-potassic  iodide,  all  of  which 
precipitate  albumins  in  the  presence  of  a  mineral  acid.  Further, 
tannic  acid,  picric  acid,  and  potassium  ferrocyanide  as  well  as  ferri- 
cyanide,  in  the  presence  of  acetic  acid,  trichloracetic  acid,  etc. 
These  various  reagents  are  used  in  from  5  to  10  per  cent,  solutions, 
after  acidifying  the  albuminous  solution  with  a  mineral  acid,  or 
acetic  acid  of  moderate  strength  as  indicated. 

The  histons  and  protamins,  which  are  more  markedly  basic  than 
the  other  classes  of  albumins,  are  precipitated  even  with  a  neutral 
or  slightly  alkaline  reaction.  But  all  precipitates  dissolve  when 
the  reaction  is  markedly  alkaline.  In  an  excess  of  the  alkaloidal 
reagent  only  the  peptones  and  some  of  the  albumoses  dissolve. 

The  precipitation  by  the  alkaloidal  reagents  is  attributed  to  the 
diamino-complcxes  of  the  albuminous  molecule,  viz.,  the  more 
markedly  basic  radicles. 

Color  Reactions. — The  color  reactions  to  be  described  are  indi- 
vidually not  peculiar  of  the  albumins,  but  merely  indicate  the 
presen(;e  of  certain  atomic  complexes  which  in  themselves  are 
capable  of  producing  the  reactions.  Collectively  they  are  charac- 
teristic, however,  and  are  of  special  interest  as  they  permit  a  partial, 
insight  into  the  structure  of  the  albuminous  molecule. 

1.  The  Biuret  Reaction. — The  test  is  conducted  as  follows  :  A 
few  c.c.  of  the  solution  to  be  examined  are  treated  with  an  excess 
of  a  concentrated  solution  of  sodium  or  potassium  hydrate,  and  then 
drop  by  drop  with  a  2  per  cent,  solution  of  copper  sulphate.  In  the 
presence  of  native  albumins  a  bluish  or  reddish-violet  color  results, 
while  with  albumoses,  peptones,  histons,  and  certain  vitellins  the 
color  is  a  pure  red.  With  larger  amounts  of  albumin  the  reaction 
is  obtained  without  difficulty ;  if  traces  only  are  present,  great  care 
must  be  had  not  to  add  too  much  of  the  copper  solution  as  other- 
wise the  blue  color  of  the  rea2:ent  mav  obscure  the  reaction.     In 


SPECIAL  REACTIONS  OF  THE  ALBUMINS.  37 

such  an  event  it  is  well  to  use  a  still  more  dilute  solution  of  the 
copper  sulphate.  Where  larger  amounts  are  present,  it  is  neces- 
sary to  add  more  of  the  reagent.  An  excess  of  the  neutral  salts 
which  are  often  present  when  the  test  is  employed  does  not  inter- 
fere with  the  reaction.  With  ammonium  sulphate,  however,  it  is 
necessary  to  use  a  large  quantity  of  the  caustic  alkali  to  bring  out 
the  color.  Should  magnesium  sulphate  be  present,  a  precipitate  of 
magnesium  hydroxide  results  oji  adding  the  alkali,  and  is  allowed 
to  settle. 

The  resulting  color,  according  to  Schiff,  is  due  to  the  formation 
of  biuret  potassium  cupric  oxide  : 

O    OH  OH     O 


/C— N  H., Cu NH2— C\ 

NH^  >NH 

"^C— NH,— K      K— NH2— C/ 

II      I  I  il 

O    OH  OH      O 

In  the  place  of  sodium  or  potassium  hydrate  other  substances 
may  also  be  used  in  tlie  test,  some  of  which  are  only  feebly  or 
indeed  scarcely  alkaline,  such  as  barium  and  calcium  liydrate,  the 
carbonates  of  the  alkalies,  ammonia,  magnesium  oxide,  trimethyl- 
amin,  coniin,  piperidin,  atropin,  etc.  Instead  of  copper  salts,  nickel 
salts  may  also  be  used ;  in  that  case  no  red  color  but  an  orange 
yellow  is  obtained. 

The  biuret  reaction  is  dependent  upon  the  presence  in  the  albuminous  molecule 
of  CH2.NH2— 

CO — NH  groups,  and  is  also  obtained  with  non-albuminous  substances,  in  which 
such  radicles  are  present — e.  g., 

CH.— XH,, 
glycinamide :  | 

CH.NH2 

CH2— NH(CH3) 

CO— NH2 

CONH2 

CH.NH2 

I 
CH, 


sarcosinamide ; 


the  diaraide  of  asparaginic  acid : 


CONHj,    and   notably  the   so-called   base   of 
Curtius:  NH2.(CH2.CO.NH)e.CH2.C02.C2Hj. 

Hofmeister  has  pointed  out  that  there  is  excellent  evidence  to 
support  the  belief  that  in  the  albuminous  molecule  the  component 
anhydrides  of  the  a-amido  acids  are  united  to  each  other  by 
— CO — NH — CH=grou]>s,  and  it  appears  that  the  same  atomic 
grouping  which  in  the  albumins  forms  the  basis  of  the  biuret  reac- 
tion is  also  the  point  of  attack  in  their  cleavage  by  the  digestive 


38  THE  ALBUMISS. 

ferments.  This  supposition  is  materially  strengthened  by  the 
interesting  observations  of  Schwarzschild  that  pure  trypsin  is 
capable  of  altering  the  base  of  Curtius  in  such  manner  that  this 
no  longer  gives  the  biuret  reaction,  while  it  is  incapable  of  splitting  off 
ammonia  from  acid  amides,  such  as  acetamide,  asparagin,  and  others. 

2.  The  Xanthoproteic  Reaction. — A  few  c.c.  of  the  solution  to  be 
tested  are  treated  with  a  few  drops  of  concentrated  nitric  acid, 
when  in  the  presence  of  most  albumins  a  white  flaky  precipitate 
develops,  which  turns  a  deep  yellow  on  heating.  With  other  forms 
the  solution  remains  clear,  but  also  turns  yellow  on  boiling.  If  in 
either  case  an  excess  of  ammonia  is  added,  a  deep-orange  color 
results,  or  with  sodium  hydrate  a  reddish  brown,  which  is  very 
characteristic. 

The  reaction  depends  upon  the  formation  of  certain  nitro  deriva- 
tives, and  is  referable  to  the  presence  in  the  albuminous  molecule 
of  a  tyrosin  group  or  of  an  indol  complex.  It  is  accordingly  not 
characteristic  of  albumins  alone,  and  can  be  obtained  with  many 
other  substances. 

3.  Millon's  Reaction. — The  reagent  is  a  solution  of  mercuric 
nitrate  containing  a  little  nitrous  acid.  It  is  prepared  by  dissolving 
a  few  grammes  of  mercuric  nitrate  in  an  amount  of  water  which  is 
just  sufficient  for  its  solution.  Any  basic  salt  that  may  be  present  is 
dissolved  with  fuming  nitric  acid,  when  a  solution  of  sodium  acetate 
is  finallv  added,  drop  by  drop,  until  the  reagent  gives  a  red  color 
on  boiling  with  a  few  drops  of  a  dilute  solution  of  phenol. 

The  test  is  conducted  by  adding  a  few  drops  of  the  solution  to  be 
examined  to  a  few  c.c.  of  the  reagent,  when  in  the  presence  of 
albumins  a  white  precipitate  forms,  which  turns  a  brick  red  on 
boiling.  If  the  substance  to  be  examined  is  a  solid,  this  is  sus- 
pended in  a  few  drops  of  water  and  treated  in  the  same  manner. 

Millon's  reaction  is  common  to  all  benzol  derivatives,  in  which 
one  hydrogen  atom  has  been  replaced  by  a  hydroxyl  group ;  it  is 
consequently  obtained  with  all  albumins  which  on  tryptic  digestion 
yield  tyrosin.  This  radicle  is  absent  in  glutin  and  in  those  albumoses 
which  contain  the  hemi-  group  (see  later),  and  these  bodies  accord- 
ingly do  not  give  the  reaction. 

4.  The  Reaction  of  Adamkiewicz. — A  particle  of  the  dry  albumin- 
ous substance,  which  should  contain  as  little  fat  as  possible,  is  dis- 
solved in  about  6  c.c.  of  glvoxylic  acid  by  the  aid  of  heat,  and 
treated  with  an  equal  volume  of  concentrated  sulphuric  acid.  Im- 
mediately or  on  boiling  red,  green,  and  violet  rings  form  at  the 
zone  of  contact  with  the  sulphuric  acid,  and  on  shaking  the  entire 
fluid  turns  violet.  At  the  same  time  it  becomes  slightly  fluorescent, 
and  on  spectroscopic  examination  gives  a  broad  band  extending  from 
yellow  to  blue. 

The  reaction  is  a  furfurol  reaction,  and  has  been  generally  ascribed 
to  the  simultaneous  presence  of  a  carbohydrate  group  and  the 
tyrosin  radicle.     In  more  recent  investigations,  however,  Hopkins 


SPECIAL  REACTIONS  OF  THE  ALBUMINS.  39 

and  Cole  have  shown  that  it  is  referable  to  the  tryptophan  complex, 
viz.,  to  skatol-amido-acetic  acid. 

5.  Liebermann's  Reaction. — The  albuminous  material  after  ex- 
traction with  hot  alcohol  and  subsequently  with  ether  (to  remove 
fats)  is  boiled  for  a  few  minutes  with  concentrated  hydrochloric 
acid  to  which  a  drop  of  concentrated  sulphuric  acid  has  been  added. 
The  albumin  passes  into  solution  and  the  fluid  assumes  a  deep-blue 
or  violet-blue  color. 

The  reaction  is  likewise  a  furfurol  reaction,  and  has  also  been 
ascribed  to  the  simidtaneous  presence  of  a  carbohydrate  and  an 
oxyphenyl  group.  Like  the  Adamkiewicz  reaction,  it  may  be 
dependent  upon  the  tryptophan  complex. 

6.  Molisch's  Reaction. — A  small  amount  of  the  albuminous  material 
is  suspended  in  about  1  c.c.  of  water  and  treated  with  a  few  drops 
of  a  15  per  cent,  alcoholic  solution  of  a-naphthol  and  with  1  or  3 
c.c.  of  concentrated  sulphuric  acid.  A  violet  color  results  (on 
standing),  which  turns  to  yellow  upon  the  addition  of  alcohol, 
ether,  or  sodium  hydrate  solution. 

Thymol  can  be  used  in  ])lace  of  the  a-naphthol ;  it  gives  a 
carmine  color,  which  on  dilution  turns  to  green. 

Like  the  two  preceding  reactions  this  one  also  is  a  furfurol  reac- 
tion ;  but  unlike  the  others  it  is  referable  exclusively  to  the  pres- 
ence of  a  carbohydrate  group.  It  is  positive  with  most  albumins, 
but  negative  with  casein. 

7.  The  Sulphur  Reaction. — A  small  amount  of  the  albuminous 
solution  is  treated  with  an  excess  of  a  concentrated  solution  of 
sodium  hydrate  and  a  few  drops  of  a  lead  acetate  solution.  On 
boiling,  according  to  the  amount  of  sulphur  present,  the  entire  fluid 
turns  a  more  or  less  marked  l^rown,  and  on  standing  a  precipitate 
of  sulphide  of  lead  results. 

The  reaction  is  referable  to  a  sulphur  group,  which  is  present  in 
the  albuminous  molecule  as  a  cystin  radicle,  as  a  thiolactic  acid 
radicle,  or  as  a  homologous  group.  As  all  albumins  contain  sulphur 
it  is  obtained  with  all,  though  to  a  more  or  less  marked  degree  (see 
page  44). 

Structural  Composition. — Our  knowledge  of  the  structural  com- 
position of  the  albumins  is  largely  the  outcome  of  a  study  of  the 
decomposition-products  obtained  by  hydrolysis,  with  boiling  mineral 
acids  and  alkalies,  by  boiling  with  water  under  })ressure,  by  diges- 
tion with  proteolytic  ferments,  both  of  animal  and  vegetable  origin  ; 
further  by  a  study  of  the  products  of  oxidation,  of  fusion  with 
caustic  alkalies,  etc.  As  a  result  it  has  been  possible  to  establish 
the  existence  in  the  albuminous  molecule  of  certain  radicles  which, 
with  few  exceptions,  are  common  to  all  forms.  The  physical  differ- 
ences of  the  various  forms  are  probably  the  outcome  of  quantitative 
variations  and  of  differences  of  chemical  union,  rather  than  of 
qualitative  composition.  These  radicles  may  be  classified  as  follows 
(Hofmeister) : 


40  THE  ALBUMINS. 

1.  Radicles  of  the  aliphatio  series. 

A.  Kadicles  containing  carbon,  nitrogen,  and  hydrogen.  Only 
one  radicle  of  this  order  has  thus  far  been  found,  but  occurs  -widely 
distributed  in  nature.     This  is  the  guanidin  remnant  CXH.XH.,. 

B.  Radicles  containing  carbon,  nitrogen,  hydrogen,  and  oxygen. 

1.  Amido-acids  : 

(a)  Mono-amido-acids. 

(a)  Radicles  of  monobasic  mono-amido-acids  (CnHj^^jNO,) ;  the 
leucin  group.  The  most  notable  radicle  of  this  order  is  leucin 
(isobutyl  amido-acetic  acid) ;  it  predominates  cpiantitatively  and  is 
present  in  all  typical  albumins.  Next  in  frequency  folloAv  a  glyco- 
coll  (amido-acetic  acid)  and  an  alanin  radicle  ;  more  rarely  the  radicle 
of  amido-valerianic  acid  and  possibly  one  of  amido-butyric  acid. 

(^9)  Radicles  of  dibasic  mono-amido-acids  (C„H.^„_,NOt) ;  the 
glutaminic  acid  group.  Of  these,  asparaginic  acid  and  glutaminic 
acid  are  probably  never  absent  in  typical  albumins,  the  latter 
predominating  in  quantity. 

(r)  Serin— C2H3(OH).(NH.).(COOH).  This  also  probably  repre- 
sents a  common  radicle  of  the  albumins. 

(6)  Di-amido-acids. 

Two  radicles  belonging  to  the  monobasic  di-amido  fatty  acids 
(C„H2„+2-'^202 — ornithin  group)  have  been  found,  viz.,  ornithin  [a-o- 
di-amino-valerianic  acid),  which  is  obtained  on  hydrolysis  with  acids 
in  combination  with  the  guanidin  remnant  in  the  form  of  arginin  ; 
and  lysin  (a-£-di-amino-capronic  acid). 

Two  other  bodies  which  may  possibly  belong  to  this  order  have 
also  been  encountered  :  the  one  a  substance  of  the  composition  of 
di-amido-acetic  acid  (a  decomposition-product  of  casein)  ;  the  other 
histidin,  CuHyXoO^,  which  is  very  commonly  found  in  association 
with  arginin. 

2.  Amido-alcohols  (hexosamins,  the  carbohydrate  group  of  the 
albumins). 

In  manyl)ut  not  in  all  typical  albumins  there  is  a  radicle  which 
on  complete  hydrolysis  appears  as  a  hexosamin  and  ajiparently 
always  as  chitosamin  (CgHnO-.NH^).  In  its  ])lace,  or  in  addition 
there  may  be  other  nitrogenous  or  non-nitrogenous  carbohydrate 
radicles.  From  the  globulins  of  the  blood-serum  Langstein  ob- 
tained glucose  and  a  sugar  of  the  character  of  fructose. 

C.  Radicles  containing  carbon,  nitrogen,  hydrogen,  oxygen,  and 
sulphur. 

The  sulphur  com])lex  of  the  albumins  is  usually  obttiined  on  hy- 
drolysis as  cystiu  (CgHi.,S.,N.,OJ,  viz.,  as  the  disulphide  of  cvstein 
(«-amido-/9-thiolactic  acid, "  CH2(SH).CH(NH„).(COOH)._  From 
serum-all)umin  and  the  keratins  Friedmann  further  obtained  the 
non-nitrogenous  /y.-thiolactic  acid,  and  suggests  that  this  also  may 
occur  in  the  albuminous  molecule  preformed.  In  addition  some 
albumins  contain  sulphur  radicles,  which  are  obtained  as  mercaptans 
or  as  a  body  which  smells  like  ethyl  sulphide. 


SPECIAL   REACTIONS  OF  THE  ALBUMINS.  41 

II.  Radicles  of  the  aromatic  series. 

A.  To  judge  from  the  constancy  witli  which  jjhenyl-alanin, 
C6H5.CH2.CH(XH2).COOH  is  encountered  among  the  hydrolytic 
decomposition-products  there  can  be  no  doubt  that  a  phenyl-amido- 
propionic  acid  radicle  is  present  in  all  albumins. 

B.  The  same  is  true  of  a  tyrosiu  radicle  (oxyphenyl-alanin), 
C,H,(OH).CH(NH2).COOH. 

III.  Heterocyclic  radicles. 

A.  Radicles  of  the  pyrrol  group.  Through  the  researches  of  E. 
Fischer  and  his  pupils  it  has  been  rendered  highly  probable  that 
an  «-pyrrolidin  carbonic  acid  radicle  (C\H-(NH).COOH)  is  repre- 
sented in  all  albumins ;  on  hydrolysis  a  corresponding  oxy-a-pyrro- 
lidin  carbonic  acid  is  apparently  also  obtained  at  the  same  time. 

B.  Radicles  of  the  indol  group.  The  indol  group  is  probably 
represented  in  the  albuminous  molecule  by  the  tryptophan  complex, 
which  Hopkins  and  Cole  have  identified  as  skatol-amido-acetic  acid 
(CuHioX.^O^).  This  radicle  is  the  mother-substance  of  indol,  skatol, 
skatol-carbonic  and  skatol-acetic  acid,  all  of  which  are  formed  during 
albuminous  putrefaction  and  in  part  also  on  fusion  with  caustic  alkali. 

Another  substance  belonging  to  this  order  is  the  skatosin  (Ci,jHig- 
N^Oo)  which  has  been  encountered  among  the  products  resulting 
from  pancreatic  autolysis,  as  also  on  prolonged  peptic  digestion  of 
serum-albumin. 

C.  Radicles  of  the  pyridin  group.  The  presence  of  a  radicle  or 
radicles  of  this  order  is  suggested  by  the  observation  of  Langstein 
that  on  long-continued  digestion  serum-albumin  yields  a  substance 
which  on  boiling  with  sodium  hydrate  gives  rise  to  the  odor  of 
pyridin.  Samueli  obtained  this  directly  on  reduction  of  nielanoi- 
dins  with  hydriodic  acid  and  zinc. 

The  presence  of  a  qninolin  radicle  is  suggested  by  the  fact  that  in 
doffs  the  ino'estion  of  albumins,  and  notably  of  a  certain  fraction  of 
albumoses  resulting  on  tryptic  digestion,  gives  rise  to  the  appearance 
in  the  urine  of  kynuric  acid  (o-oxy-/9-quinolin  carbonic  acid). 

The  presence  of  the  various  radicles  just  considered  has  l)een 
largely  established  by  a  study  of  the  decomposition-products  of  the 
albumins  which  result  on  hydrolysis,  by  means  of  boiling  mineral 
acids  and  alkalies,  proteolytic  ferments,  etc.  Other  methods  in  part 
at  least  yield  other  end-products,  M-hich,  however,  so  far  as  they 
haye  been  studied  can  readily  be  reduced  to  the  same  radicles  that 
are  furnished  by  hydrolysis. 

As  a  result  of  putrefactiye  changes  indol,  skatol,  skatol-carbonic 
acid,  and  skatol-acetic  acid  are  obtained,  all  of  which  are  derivatives  of 
the  tryptophan  complex  ;  further,  phenyl-propionic  (hydrocinnamic) 
acid,  phenvl-acetic  acid,  and  phenyl-ethylamin,  all  three  deriyatiyes 
of  phenyl-amido-propionic  acid  ;  further,  para-oxyphenyl-propionic 
(hydroparacumarie)  acid,  para-oxy]>henyl-acetic  acid,  cresol  and 
phenol,    all    derivatives   of    para-oxyphenyl-amido-propionic   acid 


42  THE  ALBUMINS. 

(tyrosin)  ;  then  a-amido-valerianic  acid  and  putrescin,  derivatives  of 
arginin  ;  cadaverin  from  lysin  ;  hydrogen  sulphide  from  cystin,  etc. 

On  oxidation  with  potassium  bichromate  and  sulphuric  acid  ben- 
zoic acid  has  been  obtained  ;  with  barium  permanganate  guanidin  is 
formed. 

On  fusion  w'ith  caustic  alkali  leucin,  tyrosin,  indol,  skatol,  hydro- 
gen sulphide,  and  methyl  mercaptan  are  quite  constantly  obtained, 
the  most  notable  being  indol  and  skatol,  whose  formation  under 
these  conditions  is  generally  regarded  as  one  of  the  most  character- 
istic reactions  of  the  albumins. 

AVith  any  method,  of  course,  products  are  also  obtained  which 
are  not  primary  components  of  the  albuminous  molecule,  but  which 
result  on  further  destruction  of  the  essential  radicles.  Such  bodies 
are  carbon  dioxide,  oxalic  acid,  formic  acid,  acetic  acid,  butyric 
acid,  isovaleric  aldehyde,  acetone,  ammonia,  hydrogen  sulphide,  etc., 

As  regards  the  manner  in  whicli  the  various  radicles  of  the  albu- 
minous molecule  are  linked  together,  our  knowledge,  while  still 
imperfect,  has  been  materially  advanced  within  recent  years  by  the 
researches  of  E.  Fischer  and  his  pupils.  As  a  result  there  is  good 
evidence  to  show  that  the  «-amido  acids  at  least  exist  in  combination 
Avith  each  other  as  so-called  polypeptides,  which  have  the  general 
structure  NH.,.(CH,.CO.NHI.CH2.COOH.      They    represent    the 

CH,.NH2 
anhvdrides  of  the  corresponding  acids  and  contain  the    | 

CO— XH 
group,  w^hich,  as  has  been  pointed  out,  is  reponsible  for  the  biuret 
reaction.     In  its  simplest  form  this  union  is  seen  in   the  binary 
compound  leucinimide,  which  would  be  a  dipeptide  in  the  sense  of 

QHg 

I 
CH 

/\ 
NH     CO 
Fischer — a  leucylleucin,  and  represented  by  the  formula  :       |       I      . 


CH 

I, 
C4H9 

That  the  albuminous  nitrogen  is  largely  ])resent  in  the  intact  mole- 
cule as  an  imido  group  is  shown  by  tiie  fact  that  only  a  small  frac- 
tion can  be  readily  split  off  as  ammonia,  while  approximately  90 
per  cent,  remains  and  on  hydrolytic  decomposition  appears  in  the 
form  of  amido-acids.  On  treating  Avith  nitrous  acid  similarly  only 
a  small  amount  of  nitrogen  is  split  ofP,  while  a  body  remains  in 
which  the  albuminous  character  is  still  preserved  to  a  large  extent, 
but  which  only  gives  an  imperfect  biuret  reaction — the  desamido- 
albumin  of  Schiff. 


SPECIAL  REACTIONS  OF  THE  ALBUMINS.  43 

Fischer's  theory  of  the  presence  in  the  albuminons  molecule  of 
the  amido-acids  in  the  form  of  polypeptides,  constructed  on  the  plan 
just  outlined,  is  strengthened  not  only  by  his  successful  synthetic 
preparation  of  dipeptides  and  tripeptides,  but  also  by  the  observation 
that  on  careful  hydr(.)lysis  of  silk  collagen  Avith  cold  hydrochloric 
acid  he  obtained  a  peptone-like  body,  from  which  trypsin  split  oif 
large  amounts  of  tyrosin,  while  a  new  body,  also  of  peptone  char- 
acter, remained  in  solution.  The  second  body  on  heating  with 
baryta  water  gave  off  ammonia  and  yielded  among  other  products 
a  dipeptide  wliich  was  apparently  glycyl-alanin. 

Still  more  recently  Fischer  and  Alderhalden  have  shown  that  on 
digestion  of  casein  with  pancreatin  a  polypeptide-like  body  remains 
which  on  total  hydrolysis  with  acids  yields  a  large  amount  of  «-pyrro- 
lidin  carbonic  acid. 

Through  the  union  of  the  amido-peptides  with  other  groups,  such 
as  the  cystin  complex,  the  glucosamin  group,  etc.,  still  more  com- 
plex radicles  result,  of  which  we  are  in  comparative  ignorance  as 
yet.  But  we  can  conceive  that  these  various  complexes  may  then 
be  further  united  to  form  still  more  complicated  radicles,  until  at 
last  the  complete  molecule  is  constructed.  In  the  typical  albumins 
we  can  distinguish  three  such  complicated  radicles,  two  of  which, 
in  conformity  with  the  nomenclature  established  by  the  Kiihne 
school,  we  may  designate  as  the  hemi-  and  the  anti-  complex  respec- 
tively, while  the  third  is  less  constant  and  differs  from  the  hemi- 
and  anti-  group  in  the  presence  of  a  carbohydrate  radicle  and  a 
larger  percentage  of  oxygen,  while  the  amount  of  nitrogen  and 
carbon  is  less  than  in  the  two  other  groups.  These  three  funda- 
mental radicles  are  represented  by  the  three  albumoses,  which 
result  primarily  on  proteolytic  digestion  (see  page  186),  viz.,  proto- 
albumose,  hetero-albumose,  and  Pick's  gluco-albnmose.  Of  these, 
proto-albumose  represents  the  hemi-  group,  hetero-albumose  the 
anti-  group,  and  gluco-albumose  the  third  complex  which  contains 
the  carbohydrate  radicle.  The  essential  points  of  difference  between 
the  hemi-  and  anti-  complex  are  the  following : 

Hemi-  group.  Anti-  group. 

Diamido-acids  in  small  amount.  Diamido-acids  in  large  amount. 

Mono-amido  acids  in  large  amount. 

Little  or  no  leucin.  Much  leucin. 

No  glycocoll.  Entire  amount  of  glycocoll  of  original 

albumin. 
Much  tyrosin.  No  tyrosin. 

Much  skatolamido-acetic    acid    (trypto-     No  skatolamido-acetic  acid. 

phan ) . 
No  carbohydrate  group.  No  carbohydrate  group. 

Contains   same    amount    of    sulphur   as     Sulphur  the  same  as  in  hemi-  group. 

original     albuminous     molecule,    but 

only  in  loosely  combined  form. 

The  anti-  group   owes  its  name  to  the  great  resistance  M'hicli  it 
offers  to  further  decomposition,  by  oxidizing  agents,  mineral  acids, 


44  THE  ALBUMINS. 

alkalies,  and  proteolytic   ferments,   notably   trypsin,  all   of  ^v]licll 
readily  decompose  the  hemi-  group  into  its  components. 

The  extent  to  which  the  hemi-,  the  anti-,  and  the  gluco-  group 
are  represented  in  the  dilierent  albumins  differs  to  a  considerable 
extent.  Casein  is  generally  regarded  as  a  pure  hemi-  body  ;  it  con- 
tains no  glycocoll,  but  more  tyrosin  than  all  true  albumins.  Colla- 
gen contains  much  glycocoll,  but  no  tyrosin,  indol,  or  tryptophan 
radicles. 

Regarding  the  intimate  structure  of  these  larger  complexes  of 
the  albuminous  molecule,  Kossel's  views  demand  special  considera- 
tion. According  to  his  idea,  these  larger  complexes  contain  a 
so-called  protone  group  as  central  nucleus,  to  which  other  prosthetic 
groups  are  united.  This  protone  group  repi'esents  the.  nucleus  of 
the  protamins,  wiiich  Kossel  regards  as  the  simplest  type  of  albu- 
min. The  protamins  in  turn  are  composed  of  one  or  more  diamido- 
acid  radicles  (see  page  54)  in  combination  with  certain  mono- 
amido-acids.  The  simplest  protamins  apparently  are  salmin 
(clupein)  and  scombrin,  in  which  of  the  diamido-acids  only  arginin 
is  present.  In  salmin  two  molecules  of  arginin  are  ])robably  united 
with  each  other  and  with  amido-valerianic  acid,  serin,  and  «-]n-rro- 
lidin  carbonic  acid. 

The  older  theory  of  Schiitzenberger,  according  to  which  all  albu- 
mins are  essentially  complex  ureids  or  oxamides,  is  untenable,  as 
there  is  no  evidence  to  show  that,  barring  the  guanidin  remnant, 
there  is  present  any  urea-yielding  group.  The  occurrence  of  an 
oxamide  or  an  oxaminic  acid  group  in  the  native  albumin  is  simi- 
larly douljtful. 

Of  the  various  radicles  which  do  not  belong  to  the  amido-acids, 
either  of  the  aliphatic  or  the  aromatic  series,  two  complexes  deserve 
especial  consideration,  namely,  those  which  represent  the  sulphur 
group  and  the  carbohydrate  grou])  of  the  albuminous  molecule. 

The  Sulplnir  Group. — Sulphur  is  present  in  all  true  all)umins  and 
in  most  albumoses.  According  to  older  views,  it  exists  in  two 
forms,  one  of  which  can  be  readily  split  off  as  hydrogen  stdjihide 
by  means  of  caustic  alkali  of  moderate  strength,  while  the  other  can 
only  be  demonstrated  after  the  complete  destruction  of  the  albumin- 
ous molecule  and  then  appears  in  the  form  of  suljihuric  acid.  It  has 
therefore  been  customary  to  speak  of  a  loosely  combined  and  a  firmly 
combined  group.  It  has  further  been  regarded  as  an  established 
fact  that  the  loosely  combined  sulphur  is  present  in  the  intact 
molecule  in  the  form  of  a  cystin  complex,  while  notliing  was  known 
of  the  form  in  which  the  so-called  oxidized  sulphur  existed. 
Morner  has  shown  that  in  certain  albumins  the  cystin-yielding 
complex  comprises  not  only  the  loosely  combined,  but  also  a  por- 
tion at  least  of  the  firmly  united  variety.  In  the  case  of  the  kera- 
tins of  horn  and  hair  it  seems  quite  likely  indeed  that  the  sulphur 
is  present  only  in  this  form.  The  shells  of  birds'  eggs,  on  the 
other  hand,  if  they  represent  a  chemical  unity  at  all,  contain  sev- 


SPECIAL  REACTIONS  OF  THE  ALBUMINS.  45 

eral  sulphur  atoms,  whicli  are  present  in  at  least  two  forms,  and 
only  one  of  which  appears  to  be  represented  by  the  cystin-yielding 
group ;  this  portion  corresponds  to  three-quarters  of  the  total 
sulphur,  while  one-quarter  is  present  in  some  other  form.  But 
even  here  the  cystiu  sulphur  includes  a  certain  portion  of  the  firmly 
combined  sulphur.  The  same  is  true  of  serum-globulin,  while  iu 
fibrinogen  and  ovalbumin  at  least  one  other  sulphur  complex 
appears  to  exist  in  addition  to  the  one  which  is  obtained  on  hydroly- 
sis as  cystin.  The  latter  in  the  case  of  fibrinogen  represents  one-half 
of  the  total  sulphur,  and  in  the  case  of  ovalbumin  only  one-third, 
while  the  remaining  two-thirds  exist  in  a  yet  unknown  form;  one- 
third  of  this  in  turn  may  under  certain  circumstances  escape  in 
volatile  form,  which,  however,  is  not  hydrogen  sulphide. 

In  the  albuminous  molecule  the  disulphide  cystin  is  the  primary 
radicle,  and  not  cystein  ;  it  hence  contains  at  least  two  molecules  of 
sulphur.  Of  other  sulphur  compounds  which  have  been  ol)tained 
from  tlie  albumins,  there  may  be  mentioned  thiolactic  acid,  thiogly- 
colic  acid,  ethyl  sulphide,  and  ethyl-  and  methyl-merkaptan.  To 
what  extent  these  bodies  occur  as  radicles  in  the  albuminous  mole- 
cule is  an  open  question.  In  the  case  of  those  albumins  iu  which 
the  sulphur  is  only  present  in  the  cystin  form  they  could  only  be 
secondary,  but  in  the  albumins  which  contain  ■  more  than  one 
sulphur  group  some  of  them  at  least  might  occur  ])reformed. 

Oxidized  sulphur — viz.,  sulphur  combined  with  oxygen — does 
not  occur  in  the  albumins. 

The  amount  of  sulphur  differs  in  the  different  albumins  (see  page 
47);  generally  speaking,  the  largest  amounts  are  found  in  the 
keratins  (2.59-5.44  ])er  cent.),  wiiile  haemoglobin  (globin)  and 
casein  contain  the  smallest  quantities. 

The  Carbohydrate  Group. — To  judge  from  recent  researches  a  car- 
bohydrate group  is  present  with  few  exceptions  in  all  albumins.  It 
characterizes  one  of  the  three  large  molecular  complexes  which  to- 
gether form  the  albuminous  molecule,  viz.,  the  gluco-complex.  As 
has  been  seen,  this  can  be  obtained  as  a  primary  product  of  pro- 
teolysis in  tlie  form  of  a  gluco-albumose,  whicli  contains  the  entire 
carbohydrate  group  of  the  original  molecule.  Notably  in  the  mucins 
and  mucoids,  but  also  in  some  of  the  native  albumins  in  the  narrower 
sense  of  the  term,  such  as  ovalbumin,  serum-albumin,  and  the  mixed 
globulins  of  the  blood-serum,  this  complex  is  obtained  on  hydrolysis 
as  an  amido-sugar,  and  usually  as  chitosamin  (glucosamin) ;  less 
commonly,  as  in  the  case  of  the  mucin  derived  from  the  frog's  so- 
called  albuminous  gland,  as  a  galactosamin.  These  bodies,  however, 
do  not  occur  preformed  in  the  albuminous  molecule,  but  as  higher 
carbohydrates,  viz.,  as  disaccharides  or  polysaccharides.  According 
to  Miiller,  the  mother-substance  of  chitosamin  is  a  polymeric  acety- 
lated  chitosamin  (Friinkel's  albamin). 

Whether  or  not  the  chitosamin  complex  is  found  in  all  albumins 
in  which  a  carbohydrate  radicle  can  be  demonstrated,  is  not  known. 


46  THE  ALBUMISS. 

In  any  event  it  is  not  the  only  carbohydrate  group  that  may  occur. 
This  is  notalily  the  case  with  the  nucleoproteids,  all  of  which  a])par- 
ently  contain  a  ])entose  group.  This  group  is  a  part  of"  the 
nucleinic  acid  radicle  of  the  nucleoproteids,  and  it  is  possible  that 
an  additional  carbohydrate  group  (perhaps  chitosamin)  may  be  con- 
tained in  the  albuminous  radicle  which  is  in  combination  with  the 
nucleinic  acid  complex  (see  page  99),  Kossel  could  demonstrate 
the  presence  of  a  hexose  also  in  the  nucleoproteid  of  the  thymus,  of 
yeast,  and  in  the  nucleinic  acid  of  the  sturgeon's  testicles,  by  isolat- 
ing kevulinic  acid  from  the  products  of  decomposition.  The  pen- 
tose obtained  from  the  nucleoproteid  of  the  pancreas  was /-xylose. 
The  amount  of  reducing  substance  (chitosamin)  which  can  be  ob- 
tained from  the  different  albumins  differs  considerably.  The  largest 
quantities  have  been  obtained  from  mucins  and  mucoids  (30-40-r 
per  cent.).  This  fact  has  led  to  the  estal)lishraent  of  a  separate  group 
of  albumins,  the  r/hicoprofeids,  which  is  distinguished  in  this  manner 
from  the  common  albumins.  This  division  is  quite  arbitrary  as  the 
latter  also  contain  a  carbohydrate  group  (glucosamin),  though  it  is 
present  in  most  of  them  in  much  smaller  amount  (serum-albumin 
0.5  per  cent.).  Crystallized  ovalbumin  with  15  per  cent,  occupies 
an  intermediate  position,  inclining  toward  the  mucoids.  Casein  is 
apparently  the  only  animal  albumin  which  is  without  a  carbohydrate 
group,  while  among  vegetable  albumins,  according  to  Osborne,  this 
is  more  frequently  the  case. 

Regarding  the  quantitative  distribution  of  the  primary  radicles  in 
the  different  albumins  our  knowledge  is  still  quite  defective,  but 
with  better  methods  is  rapidly  becoming  extended.  The  more 
important  data  have  been  embodied  in  the  accompanying  tables. 
Especially  noteworthy  is  the  extensive  quantitative  distribution  of 
leucin,  which  is  seconded  in  importance  by  pyrrolidin-carbonic  acid, 
alanin  and  phenyl-alanin,and  these  in  turn  bytyrosin  and  glycocoll. 
Quite  constant  in  the  typical  albumins  are  a-paraginic  acid  and 
glutaminic  acid,  the  latter  predominating.  Arginin  and  lysin  are 
the  most  constant  and  extensively  distributed  of  the  di-amido- 
acids ;  arginin  in  fact  has  not  as  yet  been  missed  in  any  one  of  the 
albumins.  The  same  is  true  of  the  skatol  amido-acetic  acid  com- 
plex (trvptophan).  The  notable  points  in  cr)nnection  with  the  cystin 
group  and  the  glucosamin  complex  have  been  mentioned. 


SPECIAL  REACTIONS   OF  THE  ALBUMINS  47 

Table  I. — Xitrogex  Content  op  different  Albumins. 

Nitrogen  as  Mono-amido-    Di-amido- 

ammonia.  nitrogen.  nitrogen. 

Per  cent.  Per  cent.       Per  cent. 

Seriim-albimiin  (crystallized)    ....        6.. 34  

Serum-globulin 8.90  68.28  24.95 

Egg-albumin  (crvstallizedj 8.53  67.80  21.33 

Casein    ....". 13.37  75.98  11.71 

Collagen 1.61  62.56  35.83 

Haemoglobin 6.18  63.26  23.51 

Globin 4.62  67.08  29.37 

Histon   (from  thymus)  ._ 38.40  40.50 

Proto-albumose  (from  Witte-peptone)         7.14  68.17  25.J2 

Hetero-albumose  (from  Witte-peptone)       6.45  57.40  38.93 

Table  II. — Sulphcr  Content  of  different  Albumins. 

Total  sulphur,     ^''^"sulphur^"''''^ 

Per  cent.  Per  cent. 

Serum-albumin  (crystallized) 1.73  ^  1.29 

Serum-globulin    .'....'. 0.97  ^  0.67 

Fibrinogen 1.07  ^  0.46 

Egg-albumin  (crystallized) 1.58  ^  0.43 

Haemoglobin 0.43  0.19 

Globin 0.42  0.20 

Casein 0.70  very  little. 

Keratin    (from  horn) 3.23 1  2.48 

Keratin    (from   egg-shells) 4,19  2.47 

Keratin  (from  human  hair) 5.43^  4.07 

Table  III. — Tyrosin  Content  of  different  Albumins. 

Serum-albumin 2.0  per  cent. 

Serum-globulin 3.0  " 

Egg-albumin 1.5  " 

Casein 3.0  " 

Keratin  (from  horn) 4.0  " 

Keratin  ( from  egg-shells) —  " 

Keratin  (from  human  hair) 1.6  " 

Table  IY. — Cystin  Content  of  different  Albumins. 

Serum-albumin 2.53  per  cent. 

Serum-globulin l.,51  " 

Fibrinogen 1.17  " 

Egg-albumin 0.29  " 

Keratin  (from  horn-shavings)      6.80  " 

Keratin  (from  egg-shells) 7.62  " 

Keratin  (from  hmuan  hair) 13.92  " 

Table  V. — Glycocoll  Content  of  different  Albumlns. 

Collagen 8.995  per  cent. 

Ossein 8.908  " 

Chondrin 3.794  " 

Elastin 6.595  " 

Spongin 5.012  " 

Fibroin 18.124  " 

Sericin 5.165  " 

Haemoglobin 0.465  " 

Myosin traces 

Keratin none 

^  After  deducting  sulphur  present  as  SO3. 


48  THE  A  LB  U MISS. 

Table  VI. — Distributiox  of  Argixix,  Lysix,  axd  Histidix  rs-  the  V.veious 

Albumin^. 

Histidin.  Arginin.  Lysin. 

Plt  cent.      Per  cent.  Per  ceut. 
Various  piotamins  fsalmin,  clupein, 

and  cvclupterin) 62.5-84.3C>          

Sturin  .    ." 12.90  58.20  12.00 

Histou  (from  thvmus)      1.21  14.36  7.70 

Glutin      .    .    .    ■ 9.30  5.0-6.00 

Various  vegetable  albumins     ....  0.63-1.17  4.5-11.30  0.1-5.00 

Syntonin 2.66  5.06  3.26 

Proto-albumose       0.37  8.52  7.03 

Hetero-albumose 2.20  4.90  3.50 

Deutero-albumose 1.50  7.10  6.90 

Casein 2.50  0.25  5.80 

Keratin  '  hora ) 2.25           

Molecular  Size. — Our  knowledge  of  the  molecular  .size  of  the  albu- 
mins is  still  imperfect.  This  is  owing  to  various  factors,  such  as  the 
coagulability  of  the  albumins  on  heating,  which  eliminates  the  appli- 
cation to  these  determinations  of  the  increase  in  the  boiling-point 
method ;  further,  the  present  impo.ssil)ility  of  obtaining  albumins 
entirely  free  from  .salt.s,  which  interferes  with  the  application  of  the 
method  of  determining  the  lowering  of  the  freezing-])oint,  etc. 
Attempts  to  ascertain  the  molecular  .size  from  an  analysis  of  com- 
pounds Avith  metals  have  likewise  not  proved  altogether  satisfactory'. 
The  results  which  have  l)een  obtained  are  sufficiently  definite  never- 
theless to  .show  that  the  molecular  size  is  very  great.  In  the  case 
of  the  magnesia  compound  of  a  phytovitellin  Griibler  calculated 
the  molecular  weight  as  884<S,  from  which  Bunge  deduced  the 
formula  C29.2H^iX3„0^3S2.  For  cry.«tallized  egg-albumin  Hofmeister 
established  the  formula  Cosf.Ho^XjiSoO-s,  M'hich  corresponds  to  a 
molecular  weight  of  5378.  Other  values  which  have  been  obtained 
are  6660  for  casein,  5100  for  serum-alljurain,  14,800  for  oxy- 
hseraoglobin.  For  denaturized  egg-all)umin  values  of  from  4600 
to  4700  have  been  established.  In  the  ca.se  of  the  albumoses  the 
figures  are  correspondingly  low  :  for  jjroto-albumose  about  2500, 
and  for  Siegfried's  pepsin  peptones  (which  see)  only  260-280. 

Classification  of  the  Albumins. — The  various  albumins  may  be 
divided  into  five  classes,  viz..  the  native  albumins,  the  nucle<^i-a]iju- 
mins,  the  proteids,  the  allnnninoids.  and  the  derived  albumins. 
These  can  be  subdivided  further  as  follows : 

I.    The  native  albumins  : 

1.  The    albumins    proper    (.serum-albumin,    egg-albumin, 

lactalbumin). 

2.  The  globulins  (.serum-globulin,  fibrinogen,  ovoglobulin, 

myosin,  myogen,  lactoglobulin,  various  cell-globulins, 
and  vegetable  globulins). 
•3.  The  gluco-albumins  (mucins,  mucoids,  helicoproteid). 

4.  The  keratins. 

5.  The  hi.stons. 

6.  The  protamins. 


THE  NATIVE  ALBUMINS.  49 

II.    The  nucleo-albumins  (casein,  the  vitellins,  phytovitellins,  the 
niicleo-albuinins     of     the     cell-protoplasm,     mucinous 
nucleo-albumins). 
III.    The  proteids  : 

1.  The  nucleoproteids  (compounds  of  nucleinic  acid  with 

((f)  histon,  {h)  protamin,  (c)  other  albumins. 

2.  The  haemoglobins  (comj)ounds  of  a  histon  with  haematin). 
W.  The  albuminoids  (collagen,  elastin,  spongin,  fibroin,  amyloid, 

albumoid,  pigments  which  are  derived  from  albumins). 
V.  The  derived  albumins  : 

1.  The  coagulated  albumins. 

2.  The  albuminates. 

3.  The  albumoses. 

4.  The  peptones  and  protones. 

THE  NATIVE  ALBUMINS. 

The  general  physical  and  chemical  properties  of  the  native  albu- 
mins as  a  class  have  been  described  in  the  foregoing  pages.  The 
remarks  which  follow  apply  more  particularly  to  the  various  sub- 
divisions, as  follows  : 

The  Albumins. — The  albumins,  in  the  narrower  sense  of  the  term, 
comprise  serum-albumin,  egg-albumin,  and  lactalbumin.  They  are 
soluble  in  water,  dilute  saline  solution,  and  in  dilute  acids  and  alka- 
lies. They  are  precipitated  from  their  solutions  with  greater  diffi- 
culty than  the  globulins  and  many  proteids.  Sodium  chloride  and 
magnesium  sulphate  cause  their  precipitation  only  if  the  reaction  is 
acid.  For  ammonium  sulphate  the  limits  of  precipitation  are  6.4 
and  9,  the  reaction  being  neutral ;  if  acid,  they  are  a  little  lower. 

All  the  members  of  the  group  are  coagulated  by  heat. 

One  of  the  most  interesting  properties  of  the  albumins  is  their 
ability  to  crystallize  (see  page      ). 

To  hydrolytic  decomposition  by  mineral  acids  and  ])roteolytic  fer- 
ments the  albumins  are  comjiaratively  resistant.  Notable  is  the 
large  amount  of  sulphur  of  serum-albumin  and  egg-albumin;  with 
the  exception  of  the  keratins  these  bodies  contain  more  sulphur 
(1.6  to  2.2  per  cent.)  than  any  other  proteins.  The  end-products  of 
hydrolysis  are  typical  of  the  proteins  as  a  class. 

The  Globulins. — These  comprise  the  various  globulins  of  the 
blood  (the  serum-globulins  and  fibrinogen),  ovoglobulin,  lactoglobu- 
lin,  myosin,  myogen,  various  cell-globulins  and  vegetable  globulins. 
They  are  all  soluble  in  dilute  saline  solution  and  as  a  class  insoluble 
in  water  ;  there  is  one  notable  exception,  however.  If  a  dilute 
saline  solution  of  the  common  serum-globulin  of  the  blood-plasma, 
for  example,  is  subjected  to  dialysis,  a  certain  portion  of  the  globu- 
lin is  precipitated  (euglobulin),  while  another  portion  remains  in 
solution,  and  may  be  demonstrated  by  saturating  with  magnesium 
sulphate  or  by  half-saturation   with  ammonium  sulphate   (pseudo- 

4 


50  THE  ALBUMISS. 

globulin).  With  the  exception  mentioned,  the  globulins  are  precipi- 
tated from  their  solutions  by  dialysis,  on  copious  dilution  Avith 
water,  by  acidifying  with  a  dilute  mineral  acid  or  with  acetic  acid, 
and  even  by  passing  a  stream  of  carbon  dioxide  through  the  solution. 
By  saturation  with  magnesium  sulphate  they  are  completely  })re- 
ci])itated  ;  with  sodium  chloride  only  in  part.  For  ammonium  sul- 
})hate  the  limits  are  2.9  and  4.6.  It  will  thus  be  noted  that  as  a  class 
the  globulins  can  be  readily  separated  from  the  albumins  by  half- 
saturation  with  ammonium  sulphate.  Immediately  after  precipita- 
tion by  the  various  methods  indicated  the  gkVoulins  are  still  solul)le 
in  dilute  saline  solution  ;  but  it  is  noteworthy  that  after  a  while  they 
tend  to  lose  their  solubility  and  become  coagulated;  this  also  occurs 
when  they  are  long  kept  under  water.  A  number  of  the  globulins 
show  an  acid  reaction. 

Like  the  albumins,  the  globulins  are  coagulated  by  heat ;  and  on 
hydrolysis  yield  the  same  end-products.  Their  sulj)hur  content  is 
less  than  that  of  the  albumins,  but  not  less  than  1  per  cent. 

The  Gluco-albumins. — These  com])rise  the  mucins  and  mucoids, 
the  most  notable  members  of  which  are  the  metalbumin  and  paral- 
bumin of  ovarian  cysts  (pseudomucin),  the  mucin  of  snails,  sub- 
maxillary mucin,  the  mucin  of  frog's  eggs,  and  of  sputum,  the 
mucoid  of  tendon  and  of  the  umbilical  cord,  the  chondromucoid  and 
ovomucoid,  etc.  These  bodies  are  usually  described  in  text-books 
under  a  subdivision  of  the  proteids,  viz.,  as  gJucoproteids.  Their  con- 
sideration under  that  heading  Avas  based  on  the  assumption  that  they 
represented  compound  albumins,  resulting  from  the  union  of  an  al- 
buminous radicle  with  a  special  carbohydrate  group.  Since  more 
recent  investigation  has  shown  that  this  group  is  probably  repi'e- 
sented  in  all  albumins  as  well,  and  is  merely  present  in  the  "gluco- 
proteids  "  in  especially  large  amount,  there  seems  to  be  no  adequate 
reason  for  separating  them  from  the  albumins  proper. 

The  carbohydrate  radicle  in  question  is  in  most  members  of  the 
group  obtained  in  the  form  of  chitosamin  (glucosamin),  which  in  turn 
probably  exists  in  the  intact  molecule  as  a  polysaccharide  (Frankel's 
albamin,  for  example,  see  page  45).  The  structure  of  this  com])lex 
mother-substance  is  but  little  known,  and  it  ai)pears  that  in  different 
members  of  the  group  it  may  be  different.  In  the  case  of  the 
mucins  and  mucoids  Miiller  always  obtained  the  chitosamin  in 
association  with  acetic  acid,  which  suggests  that  the  mother  sub- 
stance might  possibly  be  an  acetylated  polysaccharide.  Other 
gluco-albumins,  such  as  chondromucin,  apparently  contain  even  more 
complex  carl)ohydrate  groups,  which  yield  glucuronic  acid  besides 
chitosamin  on  hydrolytic  decomposition.  Landwehr's  animal  gum, 
which  has  figured  very  largely  as  a  decomposition-product  of  this 
order  in  the  older  text-books,  is  probably  no  unity  ;  on  decomposi- 
tion with  strong  mineral  acids  it  yields  lievulinic  acid,  leucin,  tyro- 
sin,  and  other  bodies  of  this  order. 

The  amount  of  reducing  substance  which  can  be  split  off  from 


THE  NATIVE  ALBUMINS.  51 

some  of  the  mucins  and  mucoids  is  quite  considerable.  Ovomucoid 
thus  yields  about  30  per  cent.,  submaxillary  mucin  34  per  cent.,  the 
mucin  derived  from  the  respiratory  tract  34  per  cent.,  and  the 
pseudomucin  of  ovarian  cysts  30  per  cent.  From  the  mucoid  of 
sepia  eggs  v.  Furth  obtained  36-39  per  cent. 

As  a  class  the  gluco-albumins  contain  much  less  carbon  and 
nitrogen,  but  more  oxygen,  than  the  common  albumins,  which  is 
accounted  for  by  the  large  amount  of  carbohydrate  present.^ 

According  to  Levene,  the  mucins  all  contain  the  chondroitin- 
sulphurie  acid  complex,  which  was  formerly  supposed  to  be  pecu- 
liar to  ciiondromucoid.  (Iiondroitin-sulphuric  acid  is  a  conju- 
gate sulphate,  and  on  boiling  with  dilute  hydrochloric  acid  is 
decomposed  into  sulphuric  acid  and  chondroitin.  The  latter  further 
yields  acetic  acid  and  chondrosin,  which  in  turn  supposedly  gives 
rise  to  chitosamin  and  glucuronic  acid  (Schmiedeberg).  This,  how- 
ever, has  been  disproved.  Neuberg  has  shown  that  neither  ciiitosa- 
min  nor  glucuronic  acid  results  on  hydrolysis,  but  a  tetra-oxy-amido- 
capronic  acid  and  a  carbohydrate-like  substance  of  unknown 
character.  These  decompositions  can  be  expressed  l)y  the  equa- 
tions : 

(1)  C,8H,,NOi,.S03  +  H,0  =  C,8H,,N0i,  +  ILSO, 

Choiidroitin-sul-  Chondroitin. 

phuric  acid. 

(2)  CisHj^NOu  +  SH^O  =  C,,H2iNOii  +  3CH3.COOH 
-Chondroitin.  Chondrosin.  Acetic  acid. 

(3)  C„H.,iNO„  -f  a-H,0  =  CeHi.NOe+a; 

Chondrosin,  Tetraoxy-amido-capronic 

acid. 

The  mucins  and  mucoids  are  soluble  with  great  difficulty  in 
water  and  dilute  saline  solutions,  and  are  insoluble  in  dilute  acids. 
They  possess  markedly  acid  properties  and  dissolve  in  dilute  alka- 
lies with  a  neutral  or  feebly  acid  reaction.  All  mucinous  solutions 
are  more  or  less  viscid  and  extremely  difficult  to  filter. 

The  solutions  do  not  coagulate  on  boiling,  but  on  acidifying  with 
acetic  acid  they  are  precipitated,  the  precipitate  being  insoluble  in 
an  excess  of  the  acid.  This  precipitation  by  acids  only  occurs  if 
sodium  chloride  or  other  neutral  salts  are  absent  or  present  only  in 
small  amounts.  Neutral  solutions  of  the  mucins  are  precijntated  by 
alcohol  in  the  jiresence  of  neutral  salts.  Similar  results  are  obtained 
with  some  of  the  salts  of  the  heavy  metals. 

On  heating  with  dilute  hydrochloric  acid  (2  per  cent.)  on  a 
water-bath  the  carbohydrate  group  is  split  off  and  can  be  demon- 
strated with  Folding's  test  (see  Urine). 

^  Some  of  the  analytical  results  which  have  been  obtained  are  the  following : 

Carbon. 

Mucoid  of  sepia  eggs 49.7 

Pseudomucin  (from  ovarian  cysts)     .    .    .    49.7 
Mucin  of  frog's  eargs 52.7 


Hydrogen. 

Nitrogen, 

6.96 

10.75 

6.90 

10.28 

7.10 

9.30 

52  THE  ALBUMINS. 

Certain  inncoids,  after  partial  liydrolysis  M'ith  alkalies,  <rive  a 
fine  cherry-red  color  on  treating  with  dimethyl-aniido-benzaldehyde 
and  heating  (Ehrlich). 

Whether  or  not  some  of  the  hynlogens  belong  to  the  order  of  the 
gluco-alburains  remains  to  be  seen.  Like  these,  they  contain  a 
nitrogenons  carbohydrate  radicle  which  is  probably  chitosamin. 
Such  bodies  have  been  described  especially  bv  Krukenl)erg. 
They  comprise  the  neossin  of  edible  Chinese  swallow-nests;  the 
membranin  found  in  Descemet's  membrane,  and  the  capsule  of  the 
crystalline  lens ;  the  spirographin  of  the  spirographic  membrane ; 
the  holothurian  mucin  ;  the  chondrosin  of  certain  mushrooms,  and 
others.  The  hyalin  which  is  found  in  echinococcus  cysts,  arid  the 
onuphin  of  the  tubes  of  Onuphis  fubicoh,  are  jn-obably  not  albu- 
minous. The  same  is  true  of  chitin,  which  with  other  hyalins  has 
been  found  in  the  extraskeletal  and  intraskeletal  parts  of  various 
animals. 

The  Keratins. — Formerly  the  keratins  were  classed  with  the 
albuminoids,  and  were  not  regarded  as  true  albumins.  It  has  been 
conclusively  established,  however,  that,  like  these,  they  contain  the 
guanidin  remnant,  leucin,  radicles  of  the  glutaminic  acid  group, 
ornithin  (as  arginin),  the  cystin  group,  as  well  as  an  rt:-thiolactic 
acid  nucleus  ;  further,  phenyl-alanin,  a  tyrosin  and  indol  radicle, 
as  also  a  carlx)hydrate  group.  Especially  characteristic  is  the  large 
amount  of  sulphur  (3-5  per  cent.)  which  is  obtained  in  toto  as 
cystin  on  hydrolytic  decomposition.  Tyrosin,  in  some  members  of 
the  group,  notably  in  human  hair,  is  obtained  only  in  small  amount. 
Analysis  of  some  keratins  has  given  the  following  results  : 


H 


/  C  =  50.65     H  =  6.36    N  =  17.14    S  =  5.00  O  =  20.85 
^"' \C  =  49.85     H  =  6.52     N  =  16.80    8  =  4.00  0  =  23.20 


Eee-shell  niombranes     .  C  =  49.78     H  =  6.64     N  =  16.43     S  =  4.20  O  =  22.90 
Xeiirokeratin  .        .    .    .  C  =  56.11     H  =  7.33     N  =  11.46     S  =  1.87 

The  keratins  are  the  most  im])ortant  constituents  of  the  epi- 
dermal structures  of  the  body.  They  are  found  in  the  horny  layer 
of  the  epidermis,  in  hair,  in  hoofs,  horns,  nails,  and  feathers;  further 
in  the  egg  membranes  of  birds,  of  various  reptiles  and  fishes,  etc. 
A  neurokeratin  is  found  in  the  axillary  sheath  of  the  medullated 
nerves. 

The  keratins  are  all  insoluble  in  water,  dilute  acids,  and  alka- 
lies. Strong  acids  and  alkalies  cause  their  solution,  but  at  the 
same  time  bring  about  their  disintegration.  A  solution  of  keratin 
can  therefore  not  exist. 

The  Histons. — Although  the  histons  do  not  occur  in  the  animal 
body  independently,  but  in  combination  with  certain  prosthetic 
groups,  such  as  hrematin  (in  haemoglobin)  and  nucleinic  acid  (cer- 
tain nucleoproteids),  they  are  considered  here  nevertheless,  as  they 
are  well-defined  bodies  which  are  capable  of  isolation  from  their 
pairlings  as  such.     They  comprise  the  common  histon  of  the  thynnis^ 


THE  NATIVE  ALBUMINS. 


53 


the  lymph-glands,  and  the  spleen,  the  histon  of  the  red  corpuscles 
of  the  goose,  the  salmon  and  scombron  from  the  testicles  of  the  sal- 
mon and  herring  respectively,  arbacin  from  the  spermatozoa  of 
Arbacia  pustulosa,  the  lota  histon  and  gadns  histon,  Flerolf 's  para- 
histon  and  globin,  the  albuminous  radicle  of  haemoglobin. 

According  to  Kossel,  the  histons  are  compounds  of  protamins  and 
albumins,  and  as  a  matter  of  fact  compounds  of  this  order  result 
artificially  on  treating  solutions  of  albumins  with  protamins.  The 
histons  are  in  turn  capable  of  uniting  with  albimiins,  and  may  ac- 
cordingly be  viewed  as  non-saturated  compound  albumins. 

There  are  many  points  of  similarity  between  the  histons  and  the 
protamins  (see  below).  Both  are  markedly  basic  substances,  which 
combine  with  acids  to  form  salts.  Both  form  precipitates  with  the  true 
albumins.  Both  are  precipitated  by  the  alkaloidal  reagents,  in  neu- 
tral or  alkaline  media.  Protamins  and  histons  are  both  rich  in 
basic  radicles  (diamido-acids) ;  the  histons  yield  40  per  cent,  and 
more  on  hydrolysis,  and  the  protamins  from  68  to  88  per  cent.  With 
the  exception  of  cyclopterin  the  protamins  do  not  give  Millon's  reac- 
tion and  the  histons  only  feebly. 

Histons  and  protamins  frequently  replace  each  other;  immature 
fish  sperm  thus  contains  nucleinate  of  histon,  while  in  the  mature 
material  the  corresponding  protamin  salt  is  found.  It  may,  how- 
ever, persist  as  such. 

The  elementary  composition  of  the  histons  is  different  in  different 
members  of  the  group.  Common  to  all  is  a  large  amount  of  nitro- 
gen. In  the  best  known  representative  of  the  group,  the  thymus 
histon,  elementary  analysis  has  given  the  following  results  :  C  = 
52.35  ;  H  =  7.5  ;  N  =  1 8.1  ;  and  S  =  0.62.  Its  elementary  formula, 
according  to  Bang,  is  C^-aH^jgNgiSO,,^ .  For  other  members  of  the 
group  the  following  values  have  been  obtained  : 


Salmon C  =  51.21  H  =  7.00  N  =.  17.64 

Scombron C  =  49.86  H  =  7.23  N  =  19.79 

Globin C  =  54.97  H  =  7.20  N  =  16.89 

Parahiston  (Fleroff )  .    .  C  =  51.84  H  =  7.93  N  =  17.84 


S  =  0.79  O  =  22.23 
S  =  0.42  O  =  20.52 
S  =  1.99     O  =  20.46 


The  histons  all  gave  a  violet  biuret  reaction  and  the  xanthopro- 
teic reaction,  while  Millon's  reaction  is  feeble  and  Molisch's  reaction 
absent.  A  systematic  study  of  the  hydrolytic  decomposition-prod- 
ucts has  thus  far  only  been  made  in  the  case  of  globin,  with  the 
followino^  results  : 


Per  cent. 

Tyrosin 1.33 

Alanin 4.19 

Leucin 29.04 

Glutaminic  acid 1.73 

Cystin 0.31 

Lysin 4.28 

Arginin 5.42 


Per  cent. 

Oxy-ft-pvrrolidin-carbonic  acid.      1.04 
«-Pyrrolidin-carbonic  acid  .    .      2.34 

Phenyl-alanin 4.24 

Asparaginic  acid 4.43 

Serin        0.56 

Histidin 10.96 

Tryptophan present 


54  THE  ALBUMISS. 

The  histons  are  soluble  in  water,  but  are  precipitated  on  the  addi- 
tion of  a  very  small  amount  of  ammonia  (feebly  but  distinctly  alka- 
line reaction).  In  tlie  presence  of  an  excess,  even  if  slight,  they  are 
again  dissolved,  unless  an  ammonium  salt  is  present,  in  which  case 
they  are  completely  or  almost  completely  insoluble  in  an  excess  of 
the  reagent.  This  reaction  is  very  characteristic,  but  not  altogether 
diagnostic ;  parahiston  does  not  give  it,  arbacin  only  incompletely 
so,  and  in  the  case  of  scombron  redissolution  does  not  occur  even 
though  ammonium  salts  be  absent. 

On  boiling  a  solution  of  histon  in  the  absence  of  salts  coagulation 
does  not  occur  ;  this  takes  place  if  salts  are  present,  but  it  is  note- 
worthy that  as  a  result  of  the  coagulation  denaturization  has  not 
occurred.  For  if  now  the  precipitated  histon  is  dissolved  by  the 
aid  of  an  acid  the  solution  is  neutralized  ;  the  histon  (unlike  acid 
albumin)  remains  in  solution  and  can  again  be  precipitated  on 
boiling. 

Nitric  acid  precipitates  the  histons,  but  unlike  the  common  albu- 
mins the  precipitate  dissolves  on  boiling  and  reappears  on  cooling. 
In  this  respect  they  resemble  the  albumoses. 

The  ability  of  the  histons  to  preci])itate  other  albumins  (oval- 
bumin, casein,  serum-globulin)  from  dilute  saline  solutions  has  been 
mentioned.  This  reaction,  together  with  the  behavior  of  the  histons 
toward  ammonia,  and  their  precipitation  by  the  alkaloidal  reagents 
in  neutral  media,  serve  for  their  recognition  and  identification. 

The  Protamins. — The  protamins  which  have  been  obtained  thus 
far  do  not  occur  in  the  animal  body  independently,  but  in  combina- 
tion with  certain  nucleinic  acids.  They  are  described  at  this  place 
nevertheless,  as,  like  the  histons,  they  represent  well-defined  chem- 
ical substances,  and  can  be  isolated  as  such. 

The  few  known  members  of  the  group  have  all  been  obtained 
from  the  testicles  of  various  animals  :  salmin  (ISIiescher)  from  the 
salmon,  sturin  (Kossel)  from  the  sturgeon,  clupein  (Kossel)  from 
the  herring,  scombrin  (KurajeflP)  from  the  mackerel,  cyclo]>terin 
(Kossel)  from  Cyclopterus  lumpus,  silurin  from  Silurus  glanus, 
and  accipenserin  from  Accipenser  stellatus.  Whether  or  not  Kup- 
pel's  tuberculosamin  (from  tubercle  bacilli)  also  belongs  to  this  order 
is  not  known. 

According  to  Kossel,  the  protamins  are  albumins  of  the  lowest 
order,  and  he  assumes  that  a  rndicle  of  this  kind  forms  the  nucleus 
of  all  the  more  complex  albumins.  This  assumption  primarily  was 
based  upon  the  observation  that  all  protamins  yield  certain  decom- 
position-products, which  are  also  obtained  from  the  albumins. 
These  products  are  the  diamido-acids  arginin,  lysin,  and  histidin 
(Kossel's  hexon  bases).  They  are  present  in  the  protamins  in  such 
large  amounts  (89-93.3  per  cent,  of  the  total  nitrogen)  that  Kossel 
at  first  inclined  to  the  view  that  they  alone  entered  into  the  con- 
struction of  the  ])rotamin  molecule.  Later  studies,  however, 
showed  that  still  other  radicles  are  present.     An  amido-valerianic 


^  THE  NATIVE  ALBUMINS.  55 

acid  has  thus  been  found  ;  so  also  tyrosin,  serin,  the  tryptophan 
complex,  and  in  salmin  at  least  a-pyrrolidin-carbonic  acid.  The 
protaniin  molecule  thus  proved  to  be  more  com])lex  than  Kossel  at 
first  thouf^ht,  and  other  investigators  accordingly  do  not  share  his 
views  recjardino;  the  existence  of  a  central  nucleus  of  this  order  in 
the  complex  albumins,  to  which  the  di-amido-acids  resulting  on 
hydrolysis  are  referable.  It  has  been  shown,  moreover,  that  all 
three  hexon  bases  cannot  be  obtained  from  all  albumins.  More 
recently  Kossel  has  modified  his  conception  of  the  central  protamin 
nucleus.  He  now  regards  the  simplest  ])rotamin  nucleus  as  com- 
posed of  arginin  in  combination  with  amido-valerianic  acid  and  an 
unknown  third  substance  (possibly  «-pyrrolidin-carbonic  acid).  To 
these,  in  the  higher  protamins,  still  other  radicles  are  united,  such 
as  lysin,  histidin,  and  tyrosin. 

However  this  may  be,  the  protamins  have  properties  which  war- 
rant their  classification  as  albumins.  They  all  give  the  biuret  reac- 
tion, but  neither  that  of  Millon  nor  that  of  Adamkiewicz.  They 
contain  no  sulphur.  They  are  precipitated  from  their  aqueous  solu- 
tions by  means  of  the  alkaloidal  reagents  no  matter  whether  the  reac- 
tion is  acid,  neutral,  or  alkaline.  Like  the  albumins  they  can  be 
precipitated  by  salting  (with  sodium  chloride  and  ammonium  sul- 
j)hate).  For  salmin  the  limits  of  precij)itation  with  ammonium 
sulphate  are  5.5  and  7.5.  On  heating  they  are  not  coagulated. 
Like  the  histons,  the  protamins  are  markedly  basic  and  combine 
with  acids  to  form  salts  ;  these  can  be  obtained  in  crystalline  form. 
The  sulphate  is  soluble  in  water  and  separates  out  on  cooling  or 
upon  the  addition  of  ether  as  an  oil.  With  albumins  and  the 
primary  albumoses  they  give  rise  to  precipitates  which,  according 
to  Kossel,  are  very  similar  to  the  histons. 

Tiie  protamins  are  markedly  toxic ;  they  impair  the  coagulability 
of  the  blood  and  cause  a  material  diminution  of  tlie  leucocytes. 

The  composition  of  the  various  protamins  is  expressed  in  the 

following  formulas  : 

Salmin       ^'soI^jT^nOi; 

Scombrin CfoHoo^'ieOs  (Knrajeff) 

Sturin Cs^HviXi-O^  (Ooto) 

Accipenserin CgjH-jNjgOg  (Kurajeff  ) 

Sturin ^^'iieHsgNjgO,  (Kossel) 

Cyclopterin  differs  somewhat  from  the  ordinary  ])rotamins,  and 
occupies  a  position  intermediate  between  the  histons  and  the  pro- 
tamins. It  gives  Millon's  reaction,  but  does  not  form  a  precipitate 
when  the  mixture  is  heated ;  on  cooling,  it  separates  out,  and  then 
presents  a  rose  color.  It  contains  much  less  oxygen  than  the  pro- 
tamins. Its  formula  has  not  been  ascertained  ;  elementary  analysis 
has  given  the  following  results  : 

Cyclopterin C  =  42.0  H  =  6.9  N  =  22.0 

On  hydrolytic  decomposition  the  protamins  are  first  transformed 
into  protones  (which  see). 


56  THE  'ALBUM jys. 


THE   NUCLEO-ALBUMINS   (PHOSPHOGLOBULINS). 

The  nucleo-albumins  Avere  formerly  regarded  as  a  group  of  the 
nucleuproteids,  in  Avhich  the  albuminous  component  was  united 
with  a  special  phosphorus-containing  radicle.  This  latter  was 
thought  to  be  analogous  to  the  nucleinic  radicle  of  the  true  nucleo- 
proteids ;  but  as  it  did  not  yield  the  characteristic  decomposition- 
products  of  these,  viz.,  xanthin  bases,  it  was  termed  jiaranuclein 
(Kossel)  or  pseudonuclein  (Hammarsten).  As  a  matter  of  fact,  a 
phosphorus-ctmtaining  group  can  be  obtained  from  the  nucleo- 
albumins  on  decomposition  with  pepsin-hydrochloric  acid,  but  it 
has  not  been  definitely  determined  whether  this  pseudonuclein  con- 
tains all  tiie  phosphorus  of  the  molecule.  At  present  there  is  a 
tendency  to  separate  the  nucleo-albumins  from  the  nucleoproteids, 
as  further  investigation  has  shown  that  barring  the  presence  of 
phosphorus  there  is  very  little  similarity  between  the  two.  It  has 
been  ascertained,  moreover,  that  they  probably  do  not  enter  into 
the  construction  of  the  nucleins  ;  and  as  they  resemble  the  globulins 
in  many  respects,  Cohnheim  has  suggested  that  the  term  nucleo- 
albumins  be  abandoned  and  replaced  by  phosphoglobulhis.  They 
are  nevertheless  related  to  the  nucleoproteids,  as  there  are  condi- 
tions (as  in  the  developing  organism)  under  which  the  body  unques- 
tionably constructs  its  true  nucleoproteids  from  this  source.  For 
this  reason  and  the  phosphorus  content  I  have  given  the  group  an 
independent  position  intermediate  between  the  native  albumins  and 
the  proteids. 

The  group  comprises  some  of  the  most  important  food-stuffs,  such 
as  casein,  the  vitellin  of  egg-yolk,  certain  nucleo-albumins  of  cell- 
protoplasm,  the  phytoglobulins  or  phytovitellins  of  the  leguminous 
plants,  the  ichthulin  of  fish  eggs,  etc.  Some  of  these  can  be  ob- 
tained in  crystalline  form.  All  of  them  probably  contain  iron.  Ele- 
mentary analysis  of  some  of  the  bodies  has  given  the  following 
results  : 

Casein  (Hammarsten)   .  C=5'2.90  H=7.0o  N=15.Go  S=0.758  P=0.847 

Vitellin  (birds'  eggs).   .  C=42.11  H=6.08  N=14.73  S=0.55  P=5.19  Fe=0.39 

(Bunge's  hamiatogen) 

Ichthulin  (fish  eggs)  .    .  C=53.52  H=7.71  N=15.64  S=0.41  P=0.43  Fe=0.1 

Cellular  albumin    .    .    .  C=52.37  H=6.81  N=17.23  S=1.06  P=0.42 

(snail  liver) 

Phvtovitellin(Paranuts).  C=52.43  H=7.12  N=18.1     S=0.55  P— 

Legumin  (peas)  ....  C=51.74  H=^6.90  N=18.0     S=0.42  P— 

The  ichthulin  and  the  helieoproteid  which  Hammarsten  discov- 
ered in  the  albuminous  gland  of  a  snail  (Helix  ])omatia)  were 
formerly  placed  in  a  separate  category,  the  so-called  ])h(»sphogluco- 
proteids,  as  they  contain  a  large  amount  of  reducing  substance  in 
their  molecule  ;  they  are  both,  no  doubt,  true  nucleo-albumins. 

The  nucleo-albumins  have  markedly  acid  pro])erties  and  are  almost 
insoluble  in  water.      They  form  salts  with  the  alkalies  and  ammo- 


THE  PRO  TEWS.  57 

Ilia,  and  these  are  readily  soluble  ;  on  treating  such  solutions  with 
acids  the  free  nucleo-albumins  are  again  precipitated.  The  solu- 
tions of  the  salts  are  not  coagulated  by  heat,  but  with  some  this  can 
be  effected  when  the  reaction  is  very  faintly  acid,  so  faintly  that  acid 
precipitation  does  not  occur  as  yet.  They  are  precipitated  from 
their  solutions  by  salting,  and  more  readily  so  even  than  the  globu- 
lins. 

On  peptic  digestion,  as  has  been  pointed  out,  a  pseudonuclein  is 
thrown  down  at  a  certain  ])hase  of  the  process ;  this  contains  more 
phosphorus  than  the  mother-substance.  Apparently  it  contains  a 
paranucleinic  acid  radicle.  Levene  has  isolated  a  substance  belong- 
ing to  this  order  from  birds'  eggs  (his  avivitellinic  acid),  and  there 
is  evidence  to  show  that  analogous  bodies  also  occur  in  plants.  It 
appears  quite  probable  that  the  paranucleinic  acid  of  the  egg-yolk 
represents  the  antecedent  of  the  true  nucleinic  acids  of  the  growing 
embryo.  Osborne  suggests  that  both  may  be  ethers  of  a  penta- 
hydroxyl  phosphoric  acid — H.-PO5 — or  its  first  anhydride,  HyP^O.. 

Wildenow  further  speaks  of  a  ])hosphorus-containing  substance 
which  she  obtained  from  casein,  and  which,  like  the  true  nucleinic 
acids,  precipitated  albumin.  This  has  been  confirmed  by  Salkowski, 
who  isolated  the  substance  and  gives  the  following  values  as  express- 
ing its  elementary  composition:  C  =  31.9,  H  =  4.43,  N  =  9.72, 
and  P  =  2.55. 

THE  PROTEIDS. 

The  proteids  are  conjugate  albumins  in  which  an  albuminous 
group  is  united  with  a  nuclein,  a  nucleinic  acid,  or  a  pigment  radicle. 
The  group  comprises  the  nucleoproteids,  the  nucleins,  and  the  haemo- 
globins. 

The  Nucleoproteids. — The  nucleoproteids  are  the  most  imjjor- 
tant  constituents  of  nuclear  structures  and  represent  highly  ditfer- 
entiated  albumins,  which  are  intimately  concerned  in  the  various 
manifestations  of  cell-life.  They  are  characterized  by  their  phos- 
phorus content  and  the  fact  that  on  hydrolysis  they  all  give  rise  to 
the  formation  of  xanthin  bases.  These  in  turn  are  derived  from  the 
nucleinic  acid  component  of  the  nucleoproteids,  which  in  turn  may 
be  directly  combined  with  the  primary  albuminous  radicle,  or  indi- 
rectly as  a  nuclein,  viz.,  as  an  unsaturated  nucleoproteid,  within  the 
larger  group.     These  relations  are  shown  in   the  following  schema  : 

Nucleoproteid  (first  order)  Nucleoproteid  (second  order) 


Albumin  Nucleinic  acid  .       Albumin 

Albumin      Nucleinic  acid 

From  this  it  will  be  seen  that  both  nucleins  and   nucleinic  acids 
are  derivatives  of  the  nucleoproteids.     They  can   be  isolated  from 


58  THE  ALBUMIXS. 

their  mother-substance,  but  do  not  occur  in  nature  as  such.  The 
nucleins  are  split  off  and  precipitated  on  digestion  with  pepsin- 
hydrocidoric  acid,  or  by  treating  with  acids  and  alkalies,  by  boiling 
with  water,  etc. 

Detailed  investigations  into  the  structure  of  the  complex  nucleo- 
proteids,  in  which  an  albuminous  radicle  is  in  combination  with  a 
nuclein,  are  still  wanting,  so  that  il  is  impossible  to  make  anv  defi- 
nite statements  regarding  the  allnuuiuous  group  in  either  the  nuclein 
complex  or  in  the  nucleoproteid  as  a  whole.  In  the  sim])ler  bodies, 
on  the  otiier  hand,  Avliich  have  been  obtained  from  the  testicles  of 
various  fishes  the  albuminous  component  is  apparently  always  a 
histon  or  a  protamin.  To  this  group  also  belongs  the  nucleinate  of 
histon  which  has  been  obtained  from  the  cellular  elements  of  the 
thymus  gland.  Elementary  analysis  of  some  of  these  simpler 
bodies  has  given  the  following  results : 

Nucleinate  of  arbacin  :  N  =  15.9 

Xucleinate  of  salniin :       C  =  39.85     H  =  4.86     N=  18.81     P205  =  l"-3 
10  ( C,oH5,XiP,,P,.C,6H,,X A)  -C*^54^'i A7P4 
Nucleinate  ofsalmin  Xucleinic  acid 

Nucleinate  of  clupein :       0  =  41.2      H  =  5.75    N  =  21.07     P  =  6.08    0  =  25.92 
(C3oH,,N„Oe.C«H,,N„P,0,9) 

All  or  nearly  all  nucleoproteids  contain  iron,  which,  according  to 
Kossel  and  Ascoli,  is  united  with  the  phosphorus  of  the  nucleiuic 
acid  and  not  with  the  albumin. 

More  complex  nucleoproteids  occur  in  the  pancreas,  the  thyroid, 
the  adrenal  glands,  the  mammary  glands,  the  spleen,  etc.  Some 
ferments  also  are  supposedly  of  nucleoproteid  nature. 

The  nucleo])roteids  are  all  markedly  acid  bodies.  They  are 
soluble  in  water  and  saline  solutions  and  even  more  so  in  alkalies. 
They  are  precipitated  by  acid.s,  but  dissolve  in  an  excess  (being  at 
the  same  time  decomposed).  Like  the  native  albumins,  they  are 
coagulated  by  heat  and  the  usual  reagents,  and  undergo  denaturiza- 
tion  ;  during  this  jirocess  only  the  albuminous  radicle,  however,  is 
affected.  They  can  be  precipitated  by  salting,  and  give  the  char- 
acteristic color  reactions  of  the  albumins.  Noteworthy  is  their 
behavior  toward  polarized  light.  Whereas  the  albumins  are  all  Ifevo- 
rotatory,  the  nucleo])roteids  turn  the  ])laneof  polarization  to  the  right 
(Gamgee,  Jones).  Osborne  has  shown  that  this  property  is  very 
likely  wholly  referable  to  the  nuclei nic  acid  component.  The 
degree  of  dextrorotation  may  vary  between  +37.5°  and  -|-97.9°. 

On  hydrolysis  the  nucleoproteids  yield  the  decomposition-])rod- 
ucts  of  the  albuminous  comj)onent,  besides  those  of  the  nuclcinic 
acid   radicle,  viz.,  xanthin  leases  and  phosphoric  acid. 

The  Nucleins. — The  nucleins,  as  has  been  stated,  do  not  occur  in 
nature  in  the  free  state,  but  only  in  combination  with  albumins  as 
nucleoproteids.  Their  acid  properties,  as  would  be  expected,  are  even 
more  marked  than  those  of  the  nucleoproteids,  and  they  are  .scarcely 


THE  ALBUMINOIDS.  59 

soluble  in  acids,  even  when  present  in  excess.  They  give  the  reac- 
tions of  the  albumins,  but  diifer  Iroin  these  to  a  great  extent  in  their 
elementary  composition  ;  they  contain  only  about  40  per  cent,  of  car- 
bon, but  4-7  per  cent,  of  jihosphorus.  In  gastric  juice  most  of  them 
are  insoluble,  while  they  are  readily  digested  by  trypsin.  They  are 
soluble  in  solutions  of  the  hydrates  of  the  alkalies,  less  readily  so 
in  dilute  solutions  of  the  alkaline  carbonates.  In  water  and  alcohol 
they  are  for  the  most  part  insoluble.  They  are  coagulated  by  heat, 
as  also  by  alcohol,  and  are  then  insoluble  in  solutions  of  the  alkaline 
hydrates.  Like  the  nucleoproteids  proper  the  nucleins  also  are 
dextrorotatory,  which  fact  finds  its  explanation  in  the  observation 
of  Osborne  that  this  property  is  referable  to  the  nucleinic  acid  com- 
ponent. As  would  be  expected,  the  degree  of  dextrorotation  is 
greater  in  the  nucleins  than  in  the  corresponding  nucleoproteids. 

The  Haemog"lobins. — The  group  of  the  haemoglobins  is  essen- 
tially represented  by  the  common  blood-pigment  of  vertebrate 
animals,  viz.,  haemoglobin.  Closely  related  to  it  is  the  so-called 
haemocyanin,  which  is  found  in  crabs  and  other  invertebrates,  and 
the  chlorophyll  of  plants.  The  haemoglobin  has  been  studied  with 
special  care,  and  we  now  have  a  fairly  clear  idea  of  its  structure.  In 
it  an  albuminous  radicle  belonging  to  the  histon  group,  viz.,  globin, 
is  combined  with  the  iron-containing  pigment  hrematin.  Both,  as 
also  the  haemocyanin,  will  be  considered  in  detail  in  the  section  on 
the  blood,  where  the  chemical  relation  of  the  blood-pigment  to  the 
common  respiratory  pigment  of  the  plant  will  likewise  be  pointed 
out. 

THE   ALBUMINOIDS. 

While  the  albumins  and  proteids,  which  have  been  considered  in 
the  foregoing  pages,  are  essentially  constituents  of  the  animal  cell, 
viz.,  nucleus  and  protoplasm,  the  albuminoids  or  glidinolda,  as  they 
are  also  termed,  represent  the  }>rincipal  components  of  the  intercel- 
lular structures.  They  are  thus  largely  found  in  the  supporting 
tissues  of  the  body,  viz.,  the  connective  tissue,  cartilage,  and  bone. 
In  the  nutritive  fluids,  viz.,  blood  and  lymph,  they  do  not  occur. 
The  group  comprises  the  collagens  or  glutins,  certain  vegetable 
albuminoids,  reticulin,  elastin,  the  ichthylepidin  of  fish  scales, 
various  substances  which  have  been  found  in  the  supporting  struct- 
ures of  invertebrates,  and  which  are  collectively  termed  skeletins 
(fibroin,  sericin,  spongin,  conchiolin,  cornein,  elastoidin)  and  amy- 
loid, which  is  a  ])athological  product  and  not  normally  encountered 
in  the  body.  The  keratins  have  also  been  classed  as  albuminoids 
in  the  past,  but  there  is  a  tendency  at  present  to  regard  them  as 
true  albumins  and  as  such  they  have  been  considered  in  this  work. 

The  albuminoids  are  albuminous  derivatives,  which  are  produced 
in  the  body  from  albumins  through  cellular  activity.  As  a  class 
they  do  not  contain  all  the  tyj^ical  radicles  of  the  albumins,  while 
in  some  certain  complexes  are  more  largely  represented.     The  col- 


60  THE  ALBUMJyS. 

lagens  thus  differ  from  the  true  alljuiuins  in  the  absence  of  the 
cystin  and  carbohydrate  group  ;  they  further  lack  the  tyrosin  and 
indol  group;  elastin,  fibroin,  and  sericin  lack  the  glutaminic  acid 
radicle,  etc.  Collagen,  on  the  other  hand,  contains  much  more 
glycocoll  than  the  true  albumins. 

The  nutritive  value  of  the  albuminoids  is  distinctly  less  than 
that  of  all)umins.  \o\x  has  shown  that  gelatin  (glutin,  fur  example,  is 
not  capable  of  maintaining  life.  Certain  members  of  the  group, 
moreover,  cannot  be  regarded  as  food-stuffs  in  any  sense  owing  to 
the  extreme  resistance  which  they  offer  to  most  solvents,  including 
the  digestive  fluids.  All  albuminoids  in  fact  are  insoluble  in  the 
common  solvents  of  the  albumins  (water  and  dilute  saline  solutions, 
and  for  the  most  ])art  also  in  dilute  acids  and  alkalies).  Their 
solution  involves  their  destruction. 

The  elementary  composition  of  some  of  the  more  important  mem- 
bers of  the  group  follows : 

Gelatin  (of  the  shops)  .  C  =  49.38  H  =  6.8  X  =  17.97  S  =  0.7       O  =  25.13 

Glutin  (tendon)  .  .  .  C  =  50.11  H  =  6.5  N  =  17.81  S  =  0.25  O  =  25.24 
Elastin        (ligamentum 

nuchie) C  =  54.08  H  =  7.2  N  =  16.85  S  =  0.3 

Fibroin  (silk)     .    .    .    .  C  =  48.60  H  =  6.4  ^T  =  18.89 

Amyloid       C  =  53.58  H  =  7.0  X  =  15.04  S  =  1.3 

Of  special  interest  among  the  albuminoids  is  collagen  and  its 
hydrate  (jlutin  or  f/elafin.  Solutions  of  collagen  gelatinize  on  cool- 
ing and  redissolve  on  the  apj)lication  of  heat.  The  mineral  acids, 
potassium  ferrocyanide  and  acetic  acid,  and  mo.st  mineral  salts  do 
not  precipitate  the  sub.stance  from  its  solutions.  Pure  solutions  of 
gelatin  give  the  biuret  reaction  and  the  xanthoproteic  reaction, 
while  those  of  Millon  and  Adamkiewicz  are  negative.  An  aro- 
matic complex,  however,  is  present  nevertheless,  for  on  hydrolysis 
with  mineral  acids  phenyl-alanin  may  be  obtained,  though  onlv  in 
small  amount  (0.4  per  cent.).  Noteworthy  is  the  large  amount  of 
glycocoll  (16.5  per  cent.).  Sulphur  is  present,  but  cannot  be  split 
off  as  hydrogen  sidphide  by  boiling  with  caustic  alkali. 

On  tryptic  digestion  collagen  yields  traces  of  leucin,  but  no  other 
amido-acids.  Sfdiitions  of  cartilaginous  glutin  (formerly  termed 
chondrin)  possess  characteristics  wliieh  are  different  from  those  of  the 
glutin  which  is  obtained  from  connective  ti.ssue  or  decalcified  bone. 
The.se  differences  are  not  referable  to  the  glutin  as  such,  but  to  the 
presence  of  certain  soluble  compounds  of  chondroitin-sulphuricacid. 

The  amyloid  substance,  finally,  occupies  a  unique  position  among 
the  albuminoids.  It  is  met  with  only  under  ])athologic  conditions, 
and  may  then  be  found  in  the  })aren('hyma  of  the  liver,  the  spleen, 
the  kidneys,  etc.  Ijike  the  true  albumins,  it  consists  of  ci»"^bon,  hy- 
drogen, nitrogen,  oxygen,  and  sulphur,  and  on  decompo.sition  yields 
both  leucin  and  tyrosin.  It  gives  Millon's  reaction,  that  of  Adam- 
kiewicz. and  the  xanthoproteic  reaction.  It  is  insoluble  in  water, 
alcohol,  ether,  dilute  hydrochloric  acid,  and  acetic  acid.     Concen- 


THE  DERIVED  ALBUMINS.  61 

trateil  hydrochloric  acid  and  solutions  of  the  alkaline  hydrates  cause 
its  solution,  but  at  the  same  time  transform  it  into  acid  albumin  or 
alkaline  albuminate.  The  gastric  juice,  contrary  to  what  has  been 
claimed,  likewise  causes  the  substance  to  dissolve.  Most  character- 
istic is  its  behavior  toward  iodine  and  aniline  green.  The  latter  is 
colored  red.  Dilute  aqueous  solutions  of  iodine  color  the  sul)stance 
a  brownish  red  or  a  bluish  violet,  which  passes  into  blue  on  treating 
with  sulphuric  acid.  lodomethyl-anilin  stains  the  substance  red, 
especially  after  previous  treatment  with  acetic  acid. 

THE  DERIVED  ALBUMINS. 

Fibrin. — Fibrin  occupies  a  unique  position  among  the  albumins. 
So  far  as  its  general  chemical  composition  goes,  it  is  unquestionably 
closely  related  to  the  albumins  proper.  It  contains  carbon,  hydro- 
gen, nitrogen,  oxygen,  and  sulphur  in  very  much  the  same  ])ropor- 
tion  as  the  true  albumins,  and,  like  these,  yields  the  usual  decomposi- 
tion-products on  hydrolysis.  On  the  other  hand,  fibrin  is  insoluble 
in  the  common  solvents  of  the  true  albumins,  viz.,  in  water  and 
neutral  saline  solutions,  while  acids  and  alkalies  cause  its  solution, 
but  at  the  same  time  also  its  denaturization  with  the  formation  of 
acid  albumin  or  alkaline  albuminate.  In  this  respect  fil^rin  is  closely 
related  to  the  coagulated  albumins.  It  merits  its  place  among  the 
derived  all)umins  by  reason  of  its  being  itself  a  derivative  of  a  true 
albumin,  namely,  fibrinogen,  which  is  transformed  into  fibrin  through 
the  agency  of  a  specific  ferment.  Of  the  manner  in  which  this 
transformation  is  effected  we  know  but  little.  According  to  some 
observers,  the  process  is  essentially  an  oxidation-process.  Others 
maintain  that  the  fibrin  is  present  in  the  fibrinogen  molecule  in 
combination  with  another  albuminous  group,  and  that  it  is  liberated 
under  the  influence  of  the  fibrin-ferment.  As  a  matter  of  fact  a 
certain  amount  of  another  albumin,  fibrinoglobulin,  appears  when- 
ever fibrin  itself  is  formed.  It  is  hence  a  derivative  of  a  true  albu- 
min, but  not  a  native  albumin  itself. 

The  Coagulated  Albumins. — The  coagulated  albumins  result 
from  the  albumins  proper  tlircMigh  the  influence  of  heat,  ])rolonged  ex- 
posure to  strong  alcohol,  especially  in  the  presence  of  a  neutral  salt, 
and  in  the  case  of  fibrin  at  least,  which,  as  we  have  just  seen,  is 
closely  related  to  this  group,  through  the  activity  of  a  specific  fer- 
ment. They  differ  from  the  true  albumins  in  their  extreme  resist- 
ance to  all  neutral  solvents,  and  also  to  dilute  acids  and  alkalies. 
Stronger  acids  and  alkalies  cause  their  dissolution,  with  the  simul- 
taneous formation  of  acid  albumins  or  alkaline  albuminates. 

The  Albuminates. — The  albuminates,  as  has  been  pointed  out, 
result  from  the  native  albumins  after  their  denaturization,  in  conse- 
quence of  which  their  original  characteristics  are  entirely  lost.  Aside 
from  their  quantitative  comjiosition,  thev  differ  from  each  other  only 
in  so  far  as  they  have  resulted  through  the  action  of  an  acid  or  an 


62  THE  ALBUMINS. 

alkali.  The  alkaline  albuminates  thus  contain  less  sulphur  and  less 
nitrot^en  than  the  acid  albumins,  as  a  ]K)rtion  of  the  sulphur  and  the 
so-called  amido-nitrogen  have  been  split  oif.  Both  the  acid  albu- 
mins and  the  alkaline  albuminates  are  insoluble  in  neutral  solvents, 
and  are  therefore  precipitated  from  their  solutions  on  neutralization. 
Th€\v  are  soluble,  on  the  other  hand,  in  solutions  of  the  alkaline 
hvdrates,  in  dilute  solutions  of  sodium  carbonate,  in  hydrochloric 
acid,  and  with  a  little  more  difficulty  in  strong  acetic  acid.  From 
their  acid  solutions  they  are  precipitated  by  salting  with  ammonium 
sulphate  or  sodium  chloride.  Through  the  action  of  an  alkali  acid 
albumin  can  be  transformed  into  alkaline  albuminate,  but  it  is,  of 
course,  manifest  that  the  reverse  cannot  occur.  In  the  living  body 
the  denaturization  of  all  albumins  is  effected  during  the  ]>rocess  of 
digestion,  and  invariably  precedes  the  formation  of  albumoses  and 
pejjtones. 

The  Albumoses. — The  albumoses  result  from  the  albumins 
proper,  and  also  from  the  albuminoids  and  the  albuminous  radicles 
of  the  proteids,  through  the  action  of  the  so-called  proteolytic  fer- 
ments, or  during  their  hydrolytic  decomposition  by  means  of  acids 
or  alkalies.  In  every  case  their  formation  is  preceded  by  the  dena- 
turization of  the  original  molecule. 

Collectively  the  albumoses,  which  are  derived  from  the  true  albu- 
mins in  contradistincti(m  to  those  which  are  obtained  from  the  albu- 
minoids, are  termed  ]>roteoses.  According  to  their  origin,  we  dis- 
tinguish between  globulinoses,  vitelloses,  caseoses,  myosinoses, 
keratinoses,  elastoses,  gelatoses,  etc.  During  the  process  of  diges- 
tion primary  albumoses  first  result,  which  then  give  rise  to  secondary 
albumoses,  and  these  in  turn  to  simpler  products,  Avhich  collectively 
are  termed  peptones.  Of  primary  albumoses,  at  least  three  are  known, 
viz.,  proto-albumose,  hetero-albumose,  and  gluco-albumose  (synal- 
bumose,  Hofmeister).  The  secondary  albumoses  are  designated  as 
deutero-albumoses,  and  of  these  again  several  varieties  exist  (see 
Products  of  Digestion). 

In  their  quantitative  composition  the  albumoses  do  not  differ  ma- 
terially from  the  original  albumins  ;  their  molecular  weight,  however, 
is  materially  less.  Most  likely  they  re])rcsent  depolymcrization- 
products  of  the  albumins,  and  occur  preformed  in  the  original  mole- 
cule, as  has  been  suggested  (page  43). 

The  albumoses  give  the  same  color-reactions  as  their  mother-sub- 
stances. With  the  biuret  test,  however,  instead  of  the  original  violet, 
a  beautiful  rose  color  is  obtained.  Their  final  decomposition-])ro(l- 
ucts  are  in  general  those  of  the  original  albumins  ;  there  are  certain 
differences,  however,  largely  of  a  quantitative  character,  which  are 
very  important.  These  are  shown  in  a  subsequent  table  (see 
page  202).  Noteworthy  is  the  absence  of  the  glucosamin  complex 
in  the  proto-albumose  and  hetero-albumose,  and  its  presence  in  the 
gluco-albumose  ;  further  the  large  amount  of  sulphur  in  the  thio- 
albumose  ;  then  the  presence  of  39  per  cent,  of  the  total  nitrogen,  in 


THE  DERIVED  ALBUMINS.  63 

the  form  of  hexon  bases  in  hetero-albumose,  as  compared  with  25 
per  cent,  in  the  case  ul'.proto-albumose,  etc. 

Unlike  the  albumins,  the  albumoses  are  not  entirely  indiffusible, 
and  it  appears  that  the  power  to  pass  through  animal  membranes 
increases  as  they  become  structurally  further  and  further  removed 
from  their  mother-substances. 

As  a  class  the  albumoses  are  much  more  readily  soluble  than  the 
albumins.  Most  of  them  are  soluble  in  water  or  in  dilute  saline 
solutions,  and  also  in  dilute  acids  and  alkalies.  From  their  solu- 
tions they  are  readily  precipitated  by  neutral  salts,  notably  am- 
monium sulphate,  which  precipitates  all  albumoses  when  added  to 
saturation,  the  reaction  being  slightly  acid.  ]\Iost  of  them,  indeed, 
are  thrown  down  when  the  salt  is  added  to  the  extent  of  75 
per  cent.  Each  albumose,  in  fact,  appears  to  possess  certain  special 
limits  of  precipitation  with  ammonium  sulphate  which  enables  us  to 
separate  the  individual  substances  from  each  other,  and  also  from 
other  albumins  which  may  be  present  at  the  same  time.  Zinc  sul- 
phate behaves  in  a  very  similar  manner.  Sodium  chloride  when 
added  in  substance  to  saturation  causes  a  partial  precipitation  of  the 
albumoses  from  their  neutral  solutions,  while  a  fairly  complete  sepa- 
ration is  obtained  if  to  the  saturated  fluid  is  added  a  small  amount 
of  acetic  acid  that  has  been  saturated  with  the  salt. 

Neutral  or  acid  solutions  of  albumoses  are  not  coagulated  by  heat 
nor  on  treating  with  alcohol,  although  they  are  precipitated  when 
this  is  present  in  considerable  concentration.  After  precipitation, 
however,  they  are  as  soluble  as  before,  and  in  this  respect  they  differ 
very  markedly  from  the  albumins  proper. 

Like  the  native  albumins,  the  albumoses  are  precipitated  by  nitric 
acid,  potassium  ferrocyanide  and  acetic  acid,  metaphosphoric  acid, 
phosphotungstic  acid  in  the  presence  of  hydrochloric  acid,  tannic 
acid,  picric  acid,  trichloracetic  acid,  etc.  ;  but  it  must  be  noted  that 
on  the  subsequent  application  of  heat  the  precipitate  redissolves, 
but  reappears  on  cooling.  The  same  result  is  obtained  by  treating 
a  solution  of  albumoses  with  an  equal  volume  of  a  saturated  solution 
of  sodium  chloride  and  acidifying  with  acetic  acid. 

The  Peptones. — In  conformity  with  Kiihne's  teacliings,  the  term 
peptone  has  been  used  to  designate  those  products  of  proteolytic  di- 
gestion which  can  no  longer  be  salted  out  with  ammonium  sulphate, 
either  in  neutral,  acid,  or  alkaline  media,  but  which  still  give  the 
biuret  reaction.  Products  of  this  order  are  formed  both  durinof 
peptic  and  tryptic  digestion.  Those  resulting  in  the  former  instance 
were  designated  as  ampho])eptone  on  the  assumption  that  in  it  the 
hemi-  and  the  anti-  groups  were  still  united,  and  it  was  sup- 
posed that  during  tryptic  digestion  the  hemi-  group  was  further  de- 
composed, while  the  more  resistent  antipeptone  remained  as  such. 
More  recent  researches  have  materially  modified  this  conception. 
Ktihne's  araphopeptone  and  antipeptone  have  now  been  definitely 
proved  to  be  no  unities,  and  a  hemipeptone  as  such   is   no  longer 


6-1  THE  ALBUMINS. 

reccignized.  It  has  been  shown  that  the  dilferences  between  the 
en(l-pro(hu;ts  of  pej^tic  and  tryptic  digestion  are  (juantitative,  rather 
than  qualitative,  and  that  tlie  term  peptone  comprises  a  large  num- 
ber of  hydrolytic;  decomposition-products  which  are  essentially  the 
same  as  those  which  result  from  the  albumins  on  hydrolysis  with  boil- 
ing mineral  acids  and  other  means.  Some  of  these  bodies  still  give 
the  biuret  reaction,  while  this  is  absent  in  others,  the  so-called  pcp- 
toids.  These  latter  supposedly  represent  antecedents  of  binaiy  or  at 
least  very  simple  complexes,  from  which  the  end-products  result 
directly.  Such  bodies  are  leucinimide  (C\2HoiN20.,  =  2CgH,3K02 — 
2H2O),  albamin  (a  dihexosamin  of  the  composition  C,2H24NoO<,= 
2CV,Hj3NOg — 2H2O),  arginin  (the  guauidin  radicle  plus  the  ornithin 
radicle),  glycylalanin,  etc.  E.  Fischer's  polypeptoids,  and  Siegfried's 
peptones  occupy  positions  intermediate  between  the  secondary  albu- 
moses  and  the  simpler  j)eptoids.  Some  of  the  bodies  conform  to 
Kiihne's  concept  of  antipeptone  in  so  far  as  they  energetically 
resist  further  cleavage  by  means  of  trypsin.  On  hydrolysis  these 
also  yield  the  final  end-products  of  the  less  resistent  complexes  (see 
also  page  43,  and  chapter  on  Digestive  Products). 

As  a  class  the  "  peptones "  are  crystallizable  substances,  which 
readily  diffuse  through  animal  membranes  and  possess  none  of  the 
characteristic  properties  of  the  albumins,  if  we  disregard  the  red 
biuret  reaction  which  is  obtained  with  some. 

The  term  protones  has  been  ap])lied  by  Kossel  to  products  which 
result  from  the  protamins  on  digestion  with  trypsin  and  which  stand 
intermediate  between  these  and  the  end-products.  Like  the  pro- 
tamins, they  turn  the  plane  of  polarization  to  the  left,  but  to  a  less 
extent,  and  with  cupi'ic  oxide  they  form  a  violet  compound.  From 
clupein  Goto  obtained  a  clupeon  which  corresponded  to  the  formula 
CjsHjfiNj^Og ;  in  this  80  per  cent,  of  the  total  nitrogen  was  present 
as  arginin.  A  corresponding  sturon  gave  the  formula  CggHgiNiyOg. 
As  a  class  the  protons  are  but  little  known. 


CHAPTER     III. 

THE  CAEBOHYDRATES. 

It  has  been  pointed  out  in  the  preceding  chapter  that  while 
plants  are  capable  of  effecting  from  relatively  simple  compounds  the 
synthesis  of  those  complex  albumins  which  are  found  in  their 
various  tissues  and  organs,  animals  do  not  possess  this  power, 
and  are  therefore  dependent  for  their  supply  of  nitrogen  upon  the 
albuminous  food-stuffs  that  have  been  elaborated  by  plants.  The 
carbohydrate  supply  of  animals  is  also  derived  from  plants,  but  for 
the  maintenance  of  life  it  is  not  necessary  that  this  should  be  fur- 
nished as  such,  as  animals  are  not  only  capable  of  forming  carbohy- 
drates from  the  albumins  as  (occasion  demands,  but,  as  we  shall  see 
later,  they  can  also  form  carbohydrates  directly  from  the  fats  which  are 
stored  in  their  tissues.  The  carbohydrates  cannot  therefore  be  re- 
ganled  as  essential  food-stuffs,  and  we  see,  as  a  matter  of  fact,  that 
carnivorous  animals,  at  least,  are  capable  of  existing  on  albuminous 
food  exclusively.  They  are  important,  however,  as  the  stored 
energy  which  is  thus  supplied  to  animals  represents  a  considerable 
caloric  value,  and  they  can  hence  protect  the  albumins  from  undue 
destruction.  The  importance  of  the  carbohydrates  as  food-stuffs  is 
thus  secondary,  and  they  are  totally  unable  to  take  the  place  of  the 
albumins.  All  living  matter  requires  a  definite  amount  of  nitrogen 
so  that  life  may  be  maintained,  and  if  this  is  withdrawn  death  in- 
evitably results.  It  is  to  be  noted,  however,  that  whereas  animals 
can  exist  without  carbohydrate  food,  and  whereas  the  albumins 
largely  predominate  in  their  tissues,  the  reverse  holds  good  for  plants. 
Here  the  carbohydrates  prevail,  while  the  albumins  are  much  less 
abundant.  Consequently  we  may  ex})ect  to  find  a  far  greater  diver- 
sity of  carbohydrates  in  the  vegetable  than  in  the  animal  world. 
This  is  actually  the  case.  As  it  would  lead  too  far,  in  a  work  of  this 
scope,  to  consider  all  those  carbohydrates  which  occur  in  the  vege- 
table world,  we  shall  confine  our  attention  in  the  subsequent  ]iages 
to  those  forms  which  may  be  regarded  as  common  food-stuffs,  or 
those  which  are  more  or  less  peculiar  to  the  animal  body. 

All  carbohydrates  consist  of  carbon,  hydrogen,  and  oxygen,  and 
in  most  members  of  the  group  the  elements  hydi'ogen  and  oxygen 
are  present  in  such  proportion  as  to  form  water.  In  others  this 
is  not  the  case ;  and  there  are  substances,  such  as  lactic  acid 
and  acetic  acid,  which  likewise  contain  hydrogen  and  oxygen  in 
this  proportion,  but  which  are   manifestly   not  carbohydrates.     As 

r  65 


66  THE  CARBOHYDRATES. 

there  are  no  specific  properties  peculiar  to  these  substances  as  a 
class,  it  is  impossible  to  give  an  adequate  definition  of  what  is  meant 
by  the  term  carbohydrate.  Chemically  speaking,  they  are  deriva- 
tives of  polyatomic  alcohols,  and  of  the  nature  of  aldehydes  or 
ketones.  They  are  conveniently  divided  into  monosaccharides, 
disaccharides,  and  polysaccharides.  The  disaccharides  and  poly- 
saccharides differ  from  the  monosaccharides  in  being  more  complex 
substances  and  apparently  built  up  from  the  monosaccharides 
through  a  condensation  of  monosaccharine  anhydrides  to  form  a 
double  or  a  multiple  group.  Accordingly,  on  hydrolytic  decom- 
position they  yield  two  or  more  monosaccharine  molecules  for 
every  original  molecule,  as  is  shown   below : 

(1)  CgHijOg  —   glucose,  viz.,  Ijevulose. 

(2)  C,,H,,0„    +   H,0  =   C«Hi.A   +   QHjA- 
Cane-sugar  Glucose.  Lcevulose. 

(Disaceharide.)  (>roiiosacchari(ies.) 

THE  MONOSACCHARIDES. 

According  to  the  number  of  oxygen  atoms  which  are  present  in 
the  molecule,  the  monosaccharides  can  be  divided  into  triose.s,  tet- 
roses,  pentoses,  hexoses,  hepto.ses,  octoses,  etc.  Of  these,  the  hex- 
oses  and  pentoses  only  will  be  considered,  as  the  remaining  groups 
are  not  found  in  the  animal  body. 

The  Hexoses. — The  most  important  representatives  of  the 
hexo.ses  are  glucose,  which  is  also  termed  dextrose ;  Isevulose  or 
fructose  ;  mannose  and  galactose.  Some  of  these,  such  as  glucose 
and  Isevulose,  are  found  free  in  nature,  or  they  result  as  hydrolytic 
decomposition-products  from  the  more  complex  carbohydrates  and 
related  nitrogenous  substances,  the  so-called  glucosides.  They  are 
all  derivatives  of  the  stereo-isomeric  hexatomic  alcohols  of  the 
compo.sition  CH,.OH— (CH.0H),-CH2.0H.  Of  these,  three  are 
known  to  occur  in  the  natural  state,  viz.,  sorbite,  or  glucite,  man- 
nite,  and  dulcite.  As  has  been  ]iointed  out  above,  the  mono.'^ac- 
charides  are  either  aldehydes  or  ketones,  and  we  accordingly  find 
that  glucose,  mannose,  and  galactose  represent  the  aldehydes 
(aldoses)  of  sorbite,  raanuite,  and  dulcite,  respectively,  while  licvu- 
lose  is  the  ketone  (keto.se)  of  mannite.  They  can  therefore  be 
represented  by  the  structural  formulje  : 

(1)  CH,(OH).CH(OH).CH(OH).CH(OH).CH(OH).CHO  (glucose,  mannose,  and 

galactose). 

(2)  C'Hj(OH).CHfOH).CH(OH).CH(OH).CO.CH,(OH)  (la?vulose). 

As  a  matter  of  fact  it  is  possible  to  transform  these  hexoses  into 
their  corresponding  alcohols  by  careful  reduction,  and  vice  versa. 

In  accordance  with  their  character  as  aldehydes  or  ketones,  the 
aldoses  on  oxidation  yield  oxyacids,  which  have  the  same  number 


THE  MONOSACCHARIDES.  67 

of  carbon  atoms  as  the  original  substances,  while  the  ketoses  give 
rise  to  acids  which  have  a  smaller  numl^er  of  carbon  atoms.  The 
oxyacids  which  are  derived  from  the  aldoses,  moreover,  are  either 
monobasic  or  dibasic,  according  to  the  extent  to  which  the  oxida- 
tion has  been  carried. 

These  changes  are  represented  by  the  equations  : 

(1)  CH,(OH).CH(OH).CH(OH).CH(OH).CH(OH).CHO  +  O  = 

Glucose.  CH2(0H).(CH.0H),.C00H. 

Gluconic  acid. 

(2)  CH,(OH).CH(OH).CH(OH).CH(OH).CH(OH).CHO  +  30  = 

Glucose.  COOH.(CH.OH),.COOH  +  HA 

Saecharinic  acid. 

The  acids  which  can  thus  be  obtained  from  the  aldoses  glucose, 
mannose,  and  galactose,  are  the  monobasic  acid.s — gluconic,  man- 
nonic,  and  galactouic  acid ;  and  the  dibasic  acids — saecharinic,  man- 
nosaccharinic,  and  mucinic  acid.  Of  these,  saecharinic  acid  is  of 
special  interest,  as  it  can  readily  be  transformed  into  saccharolactonic 
acid,  which  in  turn  yields  glucuronic  acid.  This  latter,  as  we  shall 
see  later,  is  found  also  in  the  animal  body.  It  is  an  aldehydic  acid, 
and  stands  midwav  between  gluconic  acid  and  .saecharinic  acid.  It 
is  represented  bv  the  formula  COOH.CH(OH).CH(OH).CH(OH). 
CH(0H).C0H: 

The  hexoses  are  colorless  and  odorless  substances  of  a  sweetish 
taste ;  they  present  a  neutral  reaction,  and  are  readily  soluble  in 
water,  with  difficulty  in  absolute  alcohol,  and  insoluble  in  ether. 
They  can  be  obtained  in  crystalline  form,  and  diffuse  through  animal 
membranes.  Some  of  them  are  dextrorotatory,  others  Isevorotatory, 
while  still  others  are  optically  inactive.  They  are  strong  reducing- 
substances,  and  for  the  most  part  fermentable  with  yeast.  Espe- 
cially interesting  further  is  the  behavior  of  the  hexoses  toward  the 
hydrazins  in  the  presence  of  acetic  acid,  with  which  they  form 
hydrazons.  These  can  be  further  transformed  into  osazons,  which 
are  very  characteristic  substances,  and  may  serve  to  distinguish  the 
various  sugars  from  each  other.  On  decomposition  with  fuming 
hydrochloric  acid  the  osazons  then  give  rise  to  the  formation  of 
oson.s — /.  €.,  keto-aldehydes,  which  can  be  further  reduced  to  ketoses. 
By  starting  with  an  aldose,  it  is  thus  possible  to  obtain  an  isomeric 
ketose.     These  changes  may  be  represented  by  the  equations  : 

(1)  CH2rOH).CH(OH).CH(OH).CH(OH).CH(OHLCHO  +  CeKs.NH.NH^^ 

Glucose  Phenvlhvdrazin. 

CH,(OH).CH(OH).CH(OH).CH(OH).CH(OH).CH:N.NH.C6H5-HjO 
Pheuylglucohydrazon. 

(2)  CH2(OH).CH(OH).CH(OH).CH(OH).CH(OH).CH:N.NH.CeH5  -^ 

C,;H5.XH.XH,= 
CH2fOH).CH(OH).CH(OH).CH(OH).C.CH.N.NH.C6H5  +  H^O  +  2H. 
Phenvlglucosazon.  || 

N.NH.C«Hs. 


68  THE  CARBOHYDRATEii. 

(3)  CH2(OH).CH(OH).CH.(OH).CH(OH).C.CH.N.NH.C6H5+2H,0+2HCl  = 

2NH3.NH.C6H6.HCI  +  CH2(0H).CH(0H).CH(OH).CH(0H).C0.C0H. 

Oson. 

(4)  CHJOH).CH(OH).CH(OH).CH.(OH).CO.CHO  +  2H= 

CH^COH).  [CH(OH)]3.COCH2(OH) 

Lavulose. 

The  same  result  may  be  reached  when  the  corresponding  osazou 
of  the  aldose  is  directly  reduced,  and  the  resulting  osamin  is  treated 
with  nitrous  acid.  The  glucosamin  thus  obtained  as  an  intermediary 
product  is  of  special  interest  in  so  far  as  it  also  results  from  the  de- 
composition of  the  hyalins  chitin  and  chondroitin.  By  oxidation  with 
bromine  glucosamin  then  yields  chitonic  acid,  from  which  the  cor- 
responding sugar,  chitose,  can  be  obtained  on  reduction.  The 
changes  which  are  here  involved  may  be  represented  by  the  equa- 
tions : 

(1)  CH2(OH).CH(OH).CH(OH).CH(OH).C.CH.N.NH.C6H5  +  H^O  +  4H  -- 

Phenylglucosazon  | 

N.NH.CeHs 
CH,(0H).CH(0H).CH(0H).CH(0H).C0.CH2(NH„)  + 

Glucosamin  C6H5.NH,.NH  +  CeHs.NH, 

Phenylhydrazin  anilin 

(2)  CHj(OH).CH(OH).CH.(OH.)CH(OH).CO.CH2(NH2)  +  HNO3  = 

Glucosamin 
HjO  +  2N  +  CH3(0H).CH(0H).CH(0H).CH(0H).C0.CH,(0H) 
Lsevulose. 

(1)  C,8H,oN,0,i,  +  4H2O  =  2CH2(OH).CH(OH).CH(OH).CH(OH). 

Chitin.  Glucosamin.  CO.CH^CNHJ  +  3CH3.COOH 

(2)  CH,(0H).CH(0H).CH(0H.)CH(0H).C0.CH2(NH,)  +40  = 

CH2(OH).CH(OH).CH(OH).CH.(OH).CH(OH).COOH  +  HNO, 
Chitonic  acid 

(3)  CH,(OH).CH(OH).CH(OH).CH(OH).CH(OH).COOH  +  2H  = 

CH2(0H).[CH{0H)],.C0H  +  H^O 

Chitose 

On  boiling  Avith  dilute  mineral  acids  the  hexoses  are  decomposed 
into  formic  acid,  Isevuliuic  acid,  and  certain  humin  substances.  With 
the  alkalies,  on  the  other  hand,  they  yield,  besides  other  products, 
also  Iffivulinic  acid  and  a  ketonic  acid  of  the  composition  CH3.CO. 
CH2.CH..COOH.  This  formation  of  Isevulinic  acid,  especially  on 
boiling  with  concentrated  hydrochloric  acid,  may  be  regarded  as 
one  of  the  characteristics  of  carboliydrates  which  are  composed  of 
6  atoms  of  carbon.  On  the  application  of  dry  heat  they  form 
so-called  caramel,  and  are  finally  carbonized.  During  the  process 
of  dry  distillation,  as  also  on  distillation  with  hydrocldoric  acid,  a 
slight  formation  of  furfurol  occurs. 

As  stated  above,  most  of  the  hexoses  are  capable  of  undergoing 
fermentation — /.  e.,  a  decompo.sition  which  is  effected  through  the 
activity  of  certain  minute  organisms.  According  to  the  character 
of  the  specific  organism  present,  we  distinguish  between  alcoholic 


THE  MONOSACCHARIDES.  69 

and  acid  fermentation,  such  as  lactic  acid,  butyric  acid,  and  acetic 
acid  fermentation.  The  former  is  bronglit  about  through  the 
influence  of  various  varieties  of  yeast,  while  the  latter  is  referable 
to  the  activity  of  certain  bacteria,  such  as  Bacterium  lactis  aerog- 
enes,  Bacillus  acidi  butyrici,  Mycoderma  aceti,  etc.  The  decom- 
positions which  are  thus  effected  may  be  represented  by  the  equa- 
tions : 

(1)  CeH,A  =  2C,H5(OH)  +  CO,. 

Ethyl  alcohol. 

(2)  CgHiA  =  2CH3— CH.OH-COOH. 
Lactic  acid. 


(3)   SCgHgOs  =  C3H7.COOH  +  2CO2  +  4H. 
Lactic  acid.      Butyric  acid. 


Of  the  hexoses,  glucose  only  i.s  found  in  the  animal  body  ;  while 
Isevulose,  raannose,  and  galactose  do  not  occur  as  such,  and  on  reach- 
ing the  liver  are  apparently  immediately  transformed,  together  with 
glucose,  into  the  polysaccharide  glycogen.  AVhether  or  not  a  trans- 
formation into  glucose  first  takes  place,  and  whether  this  can  occur 
in  the  intestinal  mucosa,  is  unknown.  The  amountof  glucose  which 
may  be  found  in  the  blood  and  lymph,  and  in  the  various  tissues  of 
the  body,  is  always  small. 

Laevulose  (fructose)  occurs  in  nature  together  with  glucose,  most 
abundantly  in  various  fruits,  the  roots  and  seeds  of  many  vegetables, 
and  also  in  honey.  It  further  results  during  the  hydrolytic  decom- 
position of  cane-sugar,  inulin,  and  other  carbohydrates.  In  the 
animal  body  it  has  been  found  in  the  blood-serum,  in  ascitic  fluid, 
and  in  pleural  effusions.  Po.ssibly  it  may  also  occur  at  times  in  the 
urine  under  pathological  conditions.  It  is  readily  soluble  in  Avater, 
and  its  aqueous  solutions,  in  contradistinction  to  common  glucose, 
are  Itevorotatory.  It  may  be  obtained  in  crystalline  form,  but  with 
difficulty.  It  is  fermentable,  and  gives  the  same  reduction-tests  as 
glucose  (which   see).       With  phenylhydrazin   Isevulose  yields   the 

C  IT 

same   osazon,  but  with   methyl-phenyl    hydrazin  ^iW  ^>N.NIl2  it 

forms  a  fructose — methyl-phenyl  o.sazon,  which  cannot  be  obtained 
either  with  glucose,  mannose,  or  glucosamin.     Its  formula  is  : 

CH2.0H.(CHOH)3.C.CH:N.N(CH3).C6H3 

II 
N.N(CH3).C6H5 

The  only  other  hexose  which  occurs  in  nature  as  such,  viz.,  sorbose, 
gives  an  oily  compound  with  the  hydrazin  in  question,  from  which 
the  corresponding  derivative  of  fructose  can  be  readily  differentiated. 
Galactose  is  formed  during  the  hydrolytic  decomposition  of 
lactose  and  many  other  carbohydrates.  It  is  also  obtained  from 
cerebriii  on  heating  with  dilute  mineral  acids.  It  is  not  so  readily 
soluble  in  water  as  glucose,  but  like  it  is  dextrorotatory.  Galactose 
crystallizes  in  needles  and  platelets  which  melt  at  168°  C.  It  is 
fermentable,  and  yields  an  osazon  which   melts  at  193°  C.     It  re- 


70  THE  CARBOHYDRATES. 

duces  an  alkaline  solution  of  cnpric  oxide,  but  to  a  less  marked 
degree  than  glucose.  On  oxidation  it  yields  first  galactonic  ac/chmd 
later  mucinio  acid. 

Glucose  will  be  considered  in  a  subsequent  chapter,  where  the 
methods  of  testing  for  the  simple  sugars  in  general,  and  also  their 
quantitative  estimation,  will  be  described. 

The  Pentoses. — The  pentoses  occur  widely  distributed  in  nature. 
In  the  animal  body  they  have  been  demonstrated  in  the  pancreas, 
the  liver,  thymus,  thyroid,  spleen,  kidneys,  salivary  glands,  brain, 
and  muscle.  The  total  amount  which  can  be  obtained  from  the  adult 
human  body  probably  represents  about  10  grammes.  The  largest 
amount  is  found  in  the  pancreas,  where  it  is  present  to  the  extent 
of  about  2.48  .per  cent,  of  the  moist  organ.  In  the  tissues  the 
pentoses  do  not  exist  in  the  free  state,  but  form  an  integral  part  of 
the  nucleoproteids,  of  which  they  are  in  a  measure  characteristic. 
Traces  of  pentoses  occur  in  the  urine  under  normal  conditions ; 
abnormally  much  larger  amounts  may  be  encountered  (see  Urine). 

The  pentoses  which  have  been  found  in  nature  are  arabinose, 
xylose,  and  rhamnose.  Neuberg  has  shown  that  of  these  /-xylose 
is  the  common  pentose  of  the  nucleoproteids  of  the  organs.  The 
optically  inactive  arabinose  occurs  in  the  urine,  where  c/-rhamnose 
has  also  been  found. 

On  prolonged  boiling  with  dilute  hydrochloric  acid  the  pentoses 
lose  water  and  form  furfurol,  viz.,  methyl-furfurol,  according  to 
the  equation  : 

C5H,oO-3H,0  =  C5HA 

Furfurol. 

This  fact  is  utilized  in  their  quantitative  estimation.  On  heat- 
ing with  hydrochloric  acid  and  phloroglucin,  or  orcin,  characteristic 
color-reactions  result  (see  Urine).  Like  the  hexoses  they  form 
compounds  with  phenylhydrazin.     They  are  not  fermentable. 

Of  special  interest  is  the  observation  that  on  fermentative  decom- 
position glucuronic  acid  passes  over  into  the  aldopentose  /-xylose  : 

COH  ( CHOH),.COOHe-*.COH  ( CH0H)3.CH,.0H 
Glucuronic  acid.  i-xylose. 

This  demonstrates  the  possible  transformation  of  a  sugar  of  the 
d-  into  one  of  the  /-series,  which  is  quite  analogous  to  what  occurs 
in  the  animal  body. 

THE   DISACCHARIDES. 

The  disaccharides  result  from  the  monosaccharides  through  a  con- 
densation of  the  anhydrides  of  two  monosaccharine  molecules,  analo- 
gous to  the  formation  of  ethers  from  alcohols.  On  hydrolytic  de- 
composition they  accordingly  yield  two  monosaccharine  molecules, 
which  represent  either  one  and  the  same  substance  or  two  isomeric 
bodies.  Some  of  the  disaccharides  occur  in  nature  as  such,  while 
others  result  from  the  decomposition  of  still  more  complex  carbo- 
hvdrates. 


THE  DISACCH ABIDES.  71 

The  most  important  members  of  the  group  are  cane-sugar  or 
saccharose,  lactose,  maltose,  and  isomaltose.  They  are  all  hexo- 
bioses — /.  e.,  they  represent  the  union  of  the  anhydrides  of  two 
hexoses,  and  can  therefore  be  represented  by  the  general  formula 
C,2H.,<,0,i.  Of  these,  cane-sugar  is  formed  through  the  union  of 
one  molecule  of  glucose  and  one  molecule  of  Isevulose  ;  lactose  from 
glucose  and  galactose;  while  maltose  contains  two  molecules  of 
glucose. 

In  their  general  properties  the  disaccharides  closely  resemble  the 
monosaccharides.  Like  these,  they  have  a  sweet  taste.  They  are 
crystallizable,  capable  of  passing  through  animal  membranes,  and 
are  optically  active.  In  certain  particulars,  however,  differences 
exist.  Lactose,  maltose,  and  isomaltose  are  thus  capable  of  reducing 
metallic  oxides  in  alkaline  solution,  and  yield  osazons  with  phenyl- 
hydrazin,  while  saccharose  does  not  react  in  this  manner. 

The  disaccharides  as  such  are  not  fermentable,  but  only  after  inver- 
sion to  monosaccharides.  It  is  true  that  a  solution  of  cane-sugar  or 
of  lactose  will  undergo  alcoholic  fermentation  when  exposed  to  the 
action  of  yeast;  but  we  now  know  that  the  yeast-cell  is  capable  of 
furnishing  certain  ferments  which  belong  to  the  class  of  the  so-called 
non-organized  ferments,  and  which  themselves  are  capable  of  bring- 
ing about  the  inversion  of  these  more  complex  carbohydrates. 
Emil  Fischer,  moreover,  has  shown  that  for  the  inversion  of  a 
special  disaccharide  a  specific  ferment  is  necessary.  As  the  various 
fermentative  agents,  however,  possess  a  varying  number  of  inverting 
ferments,  it  is  clear  that  a  certain  disaccharide  may  be  inverted  by 
one  form  of  yeast,  but  not  by  another ;  while,  on  the  other  hand, 
one  special  form  may  be  capable  of  inverting  all  forms  of  the 
disaccharides.  This  is  actually  the  case,  and  we  thus  find  that  the 
so-called  keiir  granules,  as  also  the  Bacterium  lactis,  can  invert  cane- 
sugar  as  well  as  maltose  and  lactose.  Common  yeast,  on  the  other 
hand,  inverts  only  cane  sugar  and  maltose,  while  lactose  is  not 
attacked. 

According  to  their  specific  power  of  inversion,  these  ferments  are 
spoken  of  as  invertin,  malfase,  and  lactase.  The  derivation  of  the 
two  latter  names  is,  of  course,  apparent.  The  significance  of  the 
term  invertin,  however,  is  not  so  clear.  It  has  reference  to  the 
mixture  of  glucose  and  laevulose  which  is  obtained  from  cane-sugar 
by  inversion,  and  which  was  originally  spoken  of  as  invert-sugar. 
Invertin  is  thus  a  ferment  which  inverts  cane-sugar. 

After  inversion  the  disaccharides  undergo  fermentation,  like  the 
monosaccharides,  and  here,  as  there,  we  may  distinguish  between 
alcoholic,  lactic  acid,  butyric  acid,  and  acetic  acid  fermentation. 

Cane-sugar  is  found  in  nature  most  abundantly  in  sugar-cane,  in 
the  roots  of  the  sugar  beet,  and  in  the  stems  of  certain  plants. 
The  pure  substance  is  crystalline,  and  melts  at  160°  C.  On  further 
heating  it  turns  brown  and  forms  so-called  caramel.  It  is  easily 
soluble   in  water,  and  turns  the  plane  of  polarization  to  the  right. 


72  THE  CARBOHYDRATES. 

As  stated,  it  does  not  yield  an  osazon  and  does  not  reduce  metal- 
lic oxides.  After  inversion  with  invertin  it  undero:oes  tlie  same 
fermentations  as  the  resulting  monosaccharides.  On  oxidation 
it  yields,  in  addition  to  other  substances,  saccharinic  and  oxalic 
acids. 

Maltose  does  not  occur  in  nature  as  such,  but  results  during  the 
digestion  of  starch  and  glycogen  in  tlie  alimentary  canal.  It  is  a 
crystalline  substance,  which  is  easily  soluble  in  water  and  turns  the 
plane  of  polarization  to  the  right.  AVith  plienylhydrazin  it  yields 
an  osazon — maltosazon — which  melts  at  206°  C,  It  readily  under- 
goes fermentation,  and  like  glucose  reduces  metallic  oxides  in  alka- 
line solution,  but  to  a  less  degree. 

Isomaltose  results  from  starch  through  the  action  of  a  diastatic 
ferment.  In  the  intestinal  canal  it  is  thus  found  tosrether  with 
maltose.  It  is  easily  soluble  in  water  and  turns  the  plane  of  polari- 
zation to  the  right.  Its  osazon  melts  at  150°  to  153°  C.  It  under- 
goes fermentation,  but  much  more  slowly  than  maltose. 

Lactose,  which  is  almost  exclusively  found  in  the  animal  world, 
will  be  considered  in  a  subsequent  chapter  (see  Milk). 

THE    POLYSACCHARIDES. 

The  polysaccliarides  result  from  the  monosaccharides  in  the  same 
way  as  the  disaccharides.  In  other  words,  they  represent  the 
anhydrides  of  the  monosaccharides,  of  which  many  molecides,  how- 
ever, are  condensed  to  form  the  resulting  polysacchariue  molecule. 
Their  general  formula  therefore  is  (CgHj^iOjjj.,  in  which  .t  is  a  vari- 
able factor,  but  exceeds  two.  In  many  cases  the  value  of  x  is 
unknown,  but  it  is  probably  always  large.  From  a  determination 
of  the  size  of  the  starch  molecule,  for  example,  we  may  conclude 
that  X  in  this  case  is  equivalent  to  108.  In  others,  such  as  glycogen 
and  the  dextrins,  however,  it  is  certainly  much  smaller.  In  con- 
formity with  their  structure,  the  polysaccharides  all  yield  mono- 
saccharides on  hydrolytic  decomposition.  During  this  process, 
however,  a  variable  number  of  intermediary  products  are  formed, 
which  may  themselves  be  polysaccharides,  though  of  a  lower  order, 
and  which  in  turn  yield  disaccharides  and  finally  monosaccharides. 
Starch  is  thus  first  transformed  into  erythrodextrin,  which  in  turn 
yields  achroodextrin  ;  tliis  is  further  changed  to  isomaltose,  and  then 
to  maltose,  which  finally  yields  glucose.  In  other  cases,  as  with 
glycogen,  the  disaccharides  isomaltose  and  maltose  are  formed 
directly.  Cellulose  likewise  yields  glucose  as  a  final  prochict,  while 
Isevulose  results  from  inulin,  and  mannose  from  the  so-called  reserve 
celluloses,  wliich  are  found  iu  the  cell-walls  of  many  seeds.  Galac- 
tose is  similarly  obtained  from  many  gums,  and  from  a  variety 
of  cellulose,  which  Schultze  has  termed  galactose-cellulose,  in  con- 
tradistinction to  the  mannose-cellulose  and  the  true  dextrose-cellu- 
loses.     In  many  instances,  however,  the  exact  mode  of  decomposi- 


THE  POLYSACCHARIDES.  73 

tion,  as  also  the  character  and  number  of  the  intermediary  products, 
is  but  imperfectly  understood. 

In  their  physical  and  chemical  properties  considerable  differences 
exist  between  the  polysat^charides  and  the  other  carbohydrates 
which  have  so  far  been  described.  They  are  thus  non-crystallizable 
substances  and  devoid  of  a  sweet  taste.  In  alcohol  and  ether  they 
are  insoluble.  In  water  most  of  them  are  more  or  less  soluble,  but 
as  a  class  they  are  incapable  of  diffusing  through  animal  membranes. 
From  their  solutions  they  can  be  precipitated  by  saturation  with 
neutral  salts,  and  notably  with  ammonium  sulphate.  Like  the 
monosaccharides  and  disaccharides,  they  are  optically  active,  but 
with  the  exception  of  the  dextrins  they  do  not  reduce  metallic 
oxides  in  alkaline  solution,  and  none  of  them  combine  with  phenyl- 
hydrazin  to  form  osazons.  As  such,  they  are  incapable  of  under- 
going fermentation  ;  but,  like  the  disaccharides,  they  may  be  inverted 
to  monosaccharides  through  various  ferments  or  acids,  and  can  then 
be  further  decomposed. 

Especially  important  is  their  behavior  toward  iodine,  with  which 
most  of  the  polysaccharides  combine  to  form  colored  compounds  that 
are  quite  characteristic.  Starch  is  thus  colored  blue,  glycogen  a 
mahogany  brown,  and  erythrodextrin  red. 

The  polysaccharides  which  are  used  as  food-stuffs  are  conveni- 
ently divided  into  starches,  vegetable  gums  and  dextrins,  and  cellu- 
loses. Of  these,  the  starches  are  by  far  the  most  important,  as  they 
include  not  only  vegetable  starches  and  glycogen,  but  also  give  rise 
to  formation  of  the  dextrins. 

Like  the  disaccharides,  all  these  substances  finally  give  rise  to  the 
formation  of  glycogen,  but  it  a])pears  that  they  are  previously  trans- 
formed into  glucose,  and  that  this  transformation  takes  place  in  the 
epithelial  lining  of  the  intestinal  canal. 

Starch  occurs  widely  distributed  in  the  vegetable  world,  and  con- 
stitutes the  most  important  reserve  food  of  most  of  the  higher 
plants.  It  is  found  in  the  form  of  distinct  granules,  which,  on 
microscopic  examination  exhibit  a  markt'd  concentric  striation,  and 
which  differ  in  size  and  form  in  different  plants.  The  individual 
granules  are  enclosed  in  a  capsule  of  so-called  starch  cellulose,  which 
is  insoluble  in  water,  but  which  can  be  made  to  open  by  heating  in 
the  presence  of  much  water.  The  contained  starch-granulose  can 
thus  be  obtained,  and  constitutes  the  so-called  soluble  starch,  amyhnn 
or  amylodextrin.  During  this  process  no  doubt  a  still  more  com])lex 
molecular  group  of  monosaccharine  anhydrides  is  decomposed,  but 
of  the  intermediary  products  which  are  thereby  formed,  if  any, 
nothing  is  known.  In  the  alimentary  canal  this  change  is  effected 
through  the  activity  of  certain  ferments,  which  then  further  give 
rise  to  the  formation  of  dextrin,  maltose,  isomaltose,  and  a  certain 
amount  of  glucose.  On  boiling  with  dilute  acids  glucose  is  formed, 
with  various  dextrins  as  intermediary  products.  Among  these,  as 
has  been   shown,  erythrodextrin  is  apparently  the  first  to  develop, 


74  THE  CARBOHYDRATES. 

achroodextrin  appears  later,  and  from  this  isonialtose,  maltose,  and 
glucose  are  iinally  obtained.  It  appears,  ho^vever,  that  during  the 
decompositiou  of  achroodextrin  at  least  still  other  dextrins  of  lower 
molecular  weight  are  simultaneously  formed,  which  in  turn  yield 
maltose  and  glucose.  But  finally  one  dextrin  is  obtained  which 
undergoes  no  further  change,  and  which  is  termed  maltodextrin. 

Most  characteristic  is  the  behavior  of  starch  toward  iodine,  with 
which  it  gives  an  intense  blue  color  that  disappears  on  heating,  but 
reappears  on  cooling.  In  a  solution  of  sodium  or  potassium  hydrate 
starch  swells  up  and  forms  a  paste. 

Inulin  and  lichenin,  which  also  belong  to  the  starches,  and  which 
occur  in  the  roots  of  various  composites  and  in  lichens,  respectively, 
are  insignificant  as  food-stuifs  and  need  not  be  considered. 

Glycog"en,  which  likewise  belongs  to  this  class,  and  is  known  also 
as  animal  starch,  is  largely  formed  in  the  animal  body  and  represents 
one  of  its  most  important  constituents.  It  will  be  considered  in 
detail  in  a  subsequent  chapter. 

Dextrins. — The  dextrins,  as  has  been  shown,  are  formed  from 
starch  during  its  hydrolytic  decomposition  by  means  of  ferments  or 
on  boiling  with  dilute  mineral  acids.  To  a  certain  extent  they 
result  also  when  starch  is  heated  to  a  temperature  of  from  200° 
to  210°  C.  Through  continued  decomposition  they  give  rise  to 
maltose  and  isomaltose,  and  finally  to  glucose. 

As  a  class  the  dextrins  are  easily  soluble  in  water  and  turn  the 
plane  of  polarization  to  the  right.  From  the  other  polysaccharides 
they  differ  in  their  ability  to  dissolve  cupric  hydroxide  in  alkaline 
solution.  With  erythrodextrin  iodine  strikes  a  red  color,  while 
achroodextrin  is  unaifected. 

The  so-called  vegetable  gums  and  vegetable  mucins  will  not  be 
considered,  as  they  are  of  no  importance  as  food-stuflfs.  To  this 
class  belong  the  so-called  gum  Arabic,  wood  gum,  cherry  gum,  etc., 
as  also  the  various  pectins. 

Celluloses. — As  food-stuffs  the  celluloses  are  likewise  of  second- 
ary importance.  They  are  considered,  however,  at  this  place  owing 
to  their  wide  distribution  in  the  vegetable  world,  where  they  form 
the  greater  portion  of  all  cell-envelopes.  In  the  animal  world 
they  are  likewise  encountered,  and  enter  largely  into  the  composi- 
tion of  the  external  skeleton  of  the  tunicates,  the  arthropods,  and 
some  of  the  cephalopods.  They  are  characterized  by  their  extreme 
resistance  to  the  most  divers  solvents,  and  are  indeed  soluble  only  in 
a  solution  of  cupric  hydroxide  in  strong  ammonia — the  so-called 
Schweitzer's  reagent.  From  this  solution  the  sulistance  can  be 
Cfbtained  in  amorphous  form  on  precipitation  with  acids.  Moder- 
ately concentrated  sulphuric  acid  transforms  cellulose  into  vegetable 
ami/loid,  which  is  colored  blue  by  iodine.  With  concentrated  nitric 
acid,  or  with  a  mixture  of  nitric  acid  and  concentrated  sulphuric 
acid,  it  yields  the  highly  explosive  nitrocelluloses. 

Wood  (lignin)  and  cork  are  derivatives  of  cellulose.     On  hydro- 


THE  POLYSACCHARIDES.  75 

lytic  decomposition  the  common  cellulose  yields  glucose,  while  the 
so-called  hemicelluloses  give  rise  to  galactose  or  mannose,  as  also  to 
certain  pentoses,  such  as  arabinose  and  xylose.  In  the  intestinal 
canal  a  certain  portion  of  the  ingested  cellulose  is  unquestionably 
dissolved.  The  products,  however,  to  which  it  gives  rise  are  for  the 
most  part  unknown.  Certain  micro-organisms  which  are  present  at 
the  time  bring  about  a  fermentation  of  the  substance,  during  which 
marsh  gas,  acetic  acid,  and  butyric  acid  are  formed,  but  the  greater 
portion  no  doubt  is  eliminated  in  the  feces  as  such. 


CHAPTER     IV. 

THE  FATS. 

The  origin  of  fats  in  the  animal  body  is  threefold  :  one  portion  is 
derived  from  the  fats  which  liave  been  ingested  as  food ;  another 
portion  is  formed  from  the  carbohydrates ;  while  a  third  portion 
results  from  the  decomposition  of  albumins.  As  food-stuffs  tlie  fats 
are  of  great  importance,  because  their  caloric  value  is  quite  high — 
higher  in  fact  than  that  of  the  carbohydrates  and  albumins ;  but, 
like  the  carbohydrates,  they  are  unable  to  take  the  place  of  the 
albumins.  Animals  that  are  fed  exclusively  on  fats  die  sooner  or 
later,  although  they  may  become  quite  fat  during  the  period  of  their 
special  diet.  In  the  animal  body  they  represent  a  variable  amount 
of  reserve  food,  which  is  conveniently  stored  in  the  subcutaneous 
areolar  tissue,  in  the  omentum  and  mesentery,  in  the  bone-marrow, 
etc.  In  case  of  inanition  it  is  utilized  long  before  the  tissues  of  the 
body  proper  are  attacked,  and  we  accordingly  find  that  in  persons 
who  have  died  from  wasting  diseases  every  vestige  of  fat  may  have 
disappeared,  while  the  muscular  nutrition  may  still  be  fair. 

That  portion  of  the  body  fat  which  is  derived  from  the  fats 
ingested  as  such  is  really  the  smallest  portion,  and  by  far  the  greater 
amount  results  from  the  carbohydrates.  The  manner  in  M-hich  this 
transformation  takes  place  is  unknown,  but  it  is  probable  that  the 
carbohydrates  which  are  utilized  for  this  purpose  are  first  decom- 
posed, and  then  reduced ;  and  that  the  fats  finally  result  through 
a  synthesis  of  such  reduction-products.  Such  syntheses,  however, 
cannot  at  once  be  compared  to  those  which  take  place  in  plants,  for 
here  we  have  seen  that  the  fats  can  be  formed  directly  from  water 
and  carbon  dioxide.  In  animals  this  does  not  occur,  and  we  can 
definitely  state  that  if  the  fats  are  formed  in  the  manner  indicated 
at  all,  they  result  from  very  much  more  complex  molecules. 

The  origin  of  the  fats  from  carliohydrates  can  be  demonstrated  in 
various  ways.  Dumas  and  W.  ISIilne  Edwards  have  shown  that 
bees  which  are  fed  exclusively  on  sugar  produce  three  times  as  much 
wax  as  compared  with  that  which  was  originally  ]n'esent  in  their 
bodies.  It  is  a  well-known  fact,  moreover,  that  cattle  which  are  fed 
on  nitrogenous  food  exclusively  do  not  fatten,  or  only  slightly  so  ; 
whereas  they  soon  gain  in  weight  when  a  certain  proportion  of 
carbohydrates  is  added  to  their  food. 

The  proportion  of  fiit  which  is  normally  derived  from  albumins 
is  not  very  large,  if  we  except  the  ])eriod  of  lactation  in  female 
animals,  but  its  possible  origin  from  this  source  is  undoubted. 
76 


THE  FATS.  77 

Bitches  which  are  fed  solely  on  lean  meats  continue  to  furnish  milk 
containing  an  abundance  of  butter.  Pettenkotfer  and  Voit  further 
showed  in  dogs  that  when  the  carbohydrates  remained  constant,  but 
the  albuminous  food  was  increased,  a  steady  gain  in  fat  occurred,  as 
shown  in  the  table  : 

Carbohydrates  -.i     4.  •         *  j  ^   .     .     ^ 

ingested.  Meat  ingested.  Gain  in  fat. 

379  grams.  211  grams.  24  grams. 

379      "  608      "  55      " 

379      "  1469      "  112      " 

A  further  illustration  is  had  in  the  transformation  of  the  muscular 
tissue  of  cadavers  into  so-called  adipocere,  a  substance  which  con- 
sists to  the  extent  of  97  per  cent,  of  ammonium  palmitate  with  a 
small  amount  of  stearate. 

Under  various  pathologic  conditions,  finally,  we  can  follow  with 
the  microscope  the  gradual  transformation  of  albuminous  material 
into  fat. 

All  fats  consist  of  carbon,  hydrogen,  and  oxygen.  They  are 
insoluble  in  water,  slightly  soluble  in  cold  alcohol,  while  in  hot 
alcohol,  ether,  and  benzol  they  dissolve  with  ease.  Chemically 
speaking,  they  are  neutral  compound  ethers  which  are  formed 
through  the  union  of  an  acid  with  an  alcohol  according  to  the 
equation  : 

C2H5.OH  +  CH3.COOH  =  CH3.COO.C2H5  +  H,,0. 

The  fats  which  are  principally  found  in  the  animal  world,  viz., 
palmitin,  stearin,  and  olein,  similarly  result  through  the  union  of 
their  respective  monobasic  acids  Avith  the  triatomic  alcohol  glycerin. 
This  union  is  effected  as  shown  in  the  equations : 

QH5(OH)3  +  3C,5H3,.COOH  =  C3H5(r,gH3,0,)3  +  3H,0 
Glycerin.  Palmitic  acid.  Palmitin. 

C3H5(OH)3  +  3C„H,5.COOH  =  C3H5(C,8H350,)3  +  311,0 
stearic  acid.  Stearin. 

C3H5(OH)3  +  3C„H33.COOH  =  C3H5(C,8H330,)3  +  3H,0 
Oleic  acid.  Olein. 

They  are  thus  triglycerides,  and  are  accordingly  termed  tri])al- 
mitin,  tristearin,  and  triolein.  Other  fats  have  also  been  found  in 
the  animal  world,  but  are  of  secondary  importance.  Such  fats  are 
the  so-called  cetin,  which  is  obtained  from  certain  whales,  the  myricin 
of  beeswax,  etc. 

The  animal  fat  as  a  whole  usually  represents  a  mixture  of  the 
three  triglycerides,  jxilmitin,  stearin,  and  olein,  in  variable  }n-opor- 
tions ;  the  stearin  predominating  in  the  more  solid  varieties,  while 
olein  prevails  in  the  more  liquid  fats.  In  human  fat  olein  represents 
about  670  to  800  pro  mille  of  the  total  amount. 

The  triglycerides  are  lighter  than  water ;  they  are  soluble  in 
benzol  and  ether,  and  in  hot  alcohol,  while  in  water  and  cold 
alcohol  they  are  insoluble.     They  are  non-volatile  and  burn  with  a 


78  THE  FATS. 

luminous  flame.  On  heating,  especially  in  the  presence  of  potassium 
bisulphate,  they  are  decomposed  with  the  formation  of  highly  irritat- 
ing vapors  of  an  aldehyde,  acrolein,  Avhich  in  turn  results  from 
glycerin,  according  to  the  equation  : 

CgHjlOHjs  =  C,H3.CH0  ^  2H,0. 

On  boiling  with  concentrated  alkalies,  or  through  the  influence 
of  superheated  steam,  as  also  through  certain  ferments,  the  fats  are 
decomposed  into  glycerin  and  their  respective  acids.  This  decom- 
position is  spoken  of  as  saponification,  and  the  alkaline  salts  of  the 
resulting  fatty  acids  are  accordingly  termed  "  soaps." 

On  prolonged  exposure  to  the  air,  even  in  the  absence  of  micro- 
organisms, the  fats  become  rancid — -/.  e.,  they  become  acid  and  assume 
a  most  disagreeable  odor  and  taste.  During  this  process  a  partial 
decomposition  occurs,  with  the  formation  of  glycerin  and  fatty 
acids,  which  latter  are  then  oxidized  to  certain  volatile,  offensive 
smelling  oxy-acids.  The  exact  nature  of  the  process  which  thus 
takes  place  is  not  well  understood,  but,  as  has  been  stated,  it  can 
occur  in  the  absence  of  micro-ortjanisms,  and  throuofh  the  influence 
of  light  and  air  only. 

The  fats  which  occur  in  the  animal  body  generally  present  a  more 
or  less  well-marked  yellow  or  red  color.  This  color  is  referable  to 
the  presence  of  certain  lipochromes.  These  are  compounds  which, 
like  the  fats  themselves,  are  devoid  of  nitrogen  ;  and  some  of  them 
apparently  are  hydrocarbons,  of  whose  structural  composition,  how- 
ever, nothing  is  known. 

Closely  related  to  the  fats  are  the  so-called  lecithins  and  choleste- 
rins.  The  latter  were  formerly  regarded  as  essential  food-stuffs  ; 
and  although  this  view  has  been  proved  erroneous,  they  are  never- 
theless considered  in  this  connection.  Some  of  the  lecithins,  on  the 
other  hand,  possess  a  distinct  nutritive  value. 

THE  LECITHINS. 

The  lecithins  are  ethereal  compounds  which  result  from  the  union 
of  cholin  with  glycerin-phosphoric  acid,  in  which  the  two  glycerin 
hydroxyl  groups  have  been  replaced  by  fatty  acid  radicles.  This 
union  takes  place  according  to  the  equations : 

(1)  CH2.OH  CH2.OH 

I  I 

CH.OH  +  0H.P0.(0H)2  =  CH.OH  -r  H,0 

I  I 

CH2.OH  CH,.0— PO(OH)2 

Glycerin.  Glycerfn-phosphoric  acid. 

(2)  CH2.OH  CH,.O.C„H330 

CH.OH  +  2Ci-H35.COOH=  CH.O.CigHasO  +  2H2O 

I  I 

CH2.O— PO(OH)j  CH2.O— POfOH)., 

Di-stcaryl-glycerin-phosphoric 
"acid. 


THE  LECITHINS.  79 

(3)  CH,.O.Ci8H3,0  {GK,),  CH,.O.C,8H3,0 

I  ^  I 

CH.O.C18H35O      +     N     CH2.CHO  =  CH.O.CisHaP 

CH,.0-PO(OH),  OH  CH^.O— PO.,.C.,H,\ 

Cholin.  I  \ 

OII(CH3)33^N  +  H, 

OH 

Lecithin. 

On  decomposition  of  the  lecithins  with  acids  or  alkalies  we 
accordingly  obtain  glycerin-phosphoric  acid,  fatty  acids,  and  cholin. 
At  the  same  time,  however,  another  basic  snbstance,  7ieurin,  is 
usually  found,  and  it  is  to  be  noted  that,  in  contradistinction  to 
cholin,  neurin  possesses  extremely  toxic  properties.  It  results  from 
cholin  through  the  loss  of  two  atoms  of  hydrogen  and  one  atom 
of  oxygen,  and  is  also  formed  during  bacterial  decomposition  of 
the   lecithins    in    the  presence  of  much  oxygen.     Chemically  it  is 

(CH3)3 

/ 
trimethyl-vinyl-ammonium  hydroxide,  N — CH^CHg,  while  cholin 

\ 
OH 
must  be  regarded  as  trimethyl-oxyethyl-ammonium  hydroxide. 

Another  derivative  of  cholin  which  may  be  obtained  through  the 
action  of  fuming  nitric  acid,  is  a  basic  substance  that  is  apparently 
isomeric  with  muscarin,  and,  like  this,  extremely  toxic.    Chemically 

(CH3)3 

it  may  possibly  be  represented  by  the  formula  N — CHg.COH,  and 

\ 
OH 

could  accordingly  be  regarded  as  the  aldehyde  of  oxyneurin  (tri- 

m  ethyl  glycocoll). 

The  lecithin  which  is  most  commonly  found  in  the  animal  body 
is  the  cholin  compound  of  distearyl-glycerin-phosphoric  acid  ;  other 
lecithins  can,  of  course,  also  occur,  in  which  the  glycerin  hydroxyl 
groups  have  been  replaced  by  the  radicles  of  oleic  and  palmitic  acids, 
for  example,  but  they  are  but  little  known. 

In  its  dry  state  the  common  lecithin  occurs  as  a  wax-like,  plastic 
mass,  which  is  soluble  in  alcohol  (at  40°-50°  C),  ether  (less  readily), 
chloroform,  benzol,  carbon  disul})hide,  and  in  the  fatty  oils,  while  in 
water  it  is  insoluble.  Placed  in  water  it  swells  and  becomes  ])asty, 
and  on  microscopical  examination  it  will  be  noted  that  the  substance 
occurp  in  the  form  of  peculiar  slimy  droplets  and  threads,  Avhich  are 
generally  termed  its  myelin  forms.  From  its  alcoholic  solution  it 
crystallizes  in  wart-like  masses,  which  consist  of  small  ])latelets. 

Of  special  interest  is  the  tendency  of  the  lecithins  to  combine  with 
albumins  to  form  more  or  less  stable  compounds,  which  have  l)een 
termed  lecitluilhumins.  Such  compounds  have  been  found  in  the 
mucosa  of  the  stomach,  in  the  lungs,  the  liver,  and  the  spleen.     In 


80  THE  FATS. 

the  yolk  of  eggs  it  occurs  in  combination  with  vitellin,  but  is  here 
apparently  not  closely  bound.  A  certain  similarity  thus  exists 
between  the  lecithins  and  the  nueleins  ;  both  contain  phosphorus 
in  their  molecules,  and  both  combine  with  albumins  to  form  more 
complex  substances.  The  lecithins  occur  widely  distributed  in  both 
the  animal  and  vegetable  world.  According  to  Hoppe-Seyler,  they 
are  found  in  all  cells  and  bodily  fluids.  They  are  especially  abun- 
dant in  nerve-tissue  and  also  in  the  eggs  and  semen  of  most  animals. 
Their  isolation  and  special  tests  will  be  considered  in  a  subsequent 
chapter. 

\y.  Koch  has  recently  pointed  out  the  probable  import  in  the 
life  of  the  cell  of  the  lecithins,  for  which  he  proposes  the  collective 
term  lecithanes,  and  summarizes  his  conclusions  as  follows  :  1.  In 
association  with  albumins,  in  colloid  solutions  they  furnish  the  basis 
for  the  establishment  of  the  necessary  viscosity,  by  the  ease  with 
which  they  (the  lecithanes)  are  influenced  by  ions  (Na,  Ca).  2.  They 
are  concerned  in  the  metabolism  of  the  cell,  and  in  consequence  of 
the  presence  of  the  unsaturated  fatty  acids  they  take  part  in  the 
oxygen  metabolism  and  by  means  of  their  methyl  groups  united 
to  nitrogen  in  still  other  and  unknown  reactions. 

THE  CHOLESTERINS. 

The  cholesterins  are  monatomic  alcohols  of  the  formula  CgyH^, 
OH  +  HgO,  and  occur  widely  distributed  both  in  the  vegetable  and 
the  animal  world.  They  are  especially  abundant  in  nerve-tissue 
and  in  the  bile.  In  the  gall-bladder  they  are  frequently  found  in 
the  form  of  so-called  gall-stones,  and  not  uncommonly  constitute 
the  greater  portio;i  of  their  solids.  Different  varieties  apparently 
exist,  such  as  the  common  cholesterin  of  the  concretions  just  men- 
tioned, isocholesterin  (which  has  been  obtained  from  lanolin),  phy- 
tosterin,  paracholesterin,  and  kaulosterin,  which  are  found  in 
plants.  Their  structural  composition  is  unknown.  Like  the  fats, 
they  are  insoluble  in  water,  but  soluble  in  ether,  alcohol,  and  chloro- 
form, from  which  solutions  they  may  be  obtained  either  in  the  form 
of  very  characteristic  platelets  or  as  needle-like  crystals.  In  solu- 
tions of  the  alkalies,  in  the  absence  of  alcohol,  they  are  entirely 
insoluble,  even  on  boiling,  in  which  respect  they  differ  from  the  fats. 
Like  glycerin,  cholesterin  combines  with  fatty  acids  to  form  com- 
pound ethers,  and  in  this  form  it  is  frequently  found  in  nature.  In 
wool-fat,  for  example,  it  is  thus  present  in  large  amounts,  and  from 
it  such  ethers  can  be  readily  obtained.  In  pure  form  they  constitute 
the  lanolin  of  the  shops.  These  ethers  show  a  remarkable  difference, 
as  compared  with  the  flits,  in  their  behavior  toward  water.  Of  this 
they  apparently  take  up  one-quarter  of  their  own  weight,  and  on 
stirring  give  rise  to  a  pasty,  frothy   mass. 

From  their  ethereal  compounds  cholesterin  can  readily  be  sepa- 
rated by   treating   with  diacetic- ethyl   ether,  which    dissolves    thf 


THE  CHOLESTERINS.  81 

uholesterin  and  leaves  the  ethers  behind.  Their  special  tests,  and 
also  their  mode  of  isolation,  will  be  taken  up  in  a  subsequent 
chapter. 

After  having  thus  studied  the  three  great  classes  of  food-stuffs 
which  plants  are  capable  of  elaborating  from  water,  carbon  dioxide, 
and  certain  mineral  salts,  and  which  are  also  represented  in  the 
animal  body,  we  shall  now  ])roceed  to  a  similar  survey  of  the  natural 
decomposition-products  of  these  substances  which  are  formed  during 
their  passage  through  the  animal  body,  and  which  are  of  more  or  less 
interest  in  indicating  the  manner  in  which  these  decompositions 
are  eifected.  Like  the  substances  that  have  already  been  con- 
sidered, these  products  will  be  taken  up  only  in  a  general  way  at 
first ;  while  their  special  study,  as  well  as  their  methods  of  isolation 
and  quantitative  estimation,  will  be  considered  in  succeeding  chapters, 
in  connection  with  the  chemical  study  of  those  tissues  in  which  they 
are  principally  encountered.  At  this  place  I  wish  only  to  impress 
general  facts  upon  the  mind  of  the  student,  so  that  he  will  be  pre- 
pared to  understand  the  composite  chemical  structure  of  the  various 
tissues  and  organs  of  the  body,  as  will  be  described  later. 


CHAPTER   Y. 

THE   XITROGEXOUS   DERIVATIVES   OF  THE   ALBUMINS. 

THE   DIAMIDO-ACIDS. 

The  dianiido-acids  in  question  are  arginin,  lysin,  and  liistidiu, 
which  Kossel  collectively  terms  the  hexon  base-n.  They  result  on 
hydrolytic  decomposition  of  the  albumins,  inclusive  of  the  prota- 
mins,  as  has  l)een  shown  (page  40).  The  free  bases,  like  tht  ■^  ro- 
tamins,  are  lsevf)rotatory,  while  their  salts,  which  result  through 
their  union  with  acids  and  salts  of  the  heavy  metals,  are  dextro- 
rotatory.    These  salts  can  be  obtained  in  crystalline  form. 

Arginin. — On  hydrolytic  decomposition  arginin  yields  urea  and 
ornithin.  The  substance  is  therefore  regarded  as  a  guanidin  deriva- 
tive, similar  to  kreatin,  in  which  one  amido-group  has  been  replaced 
by  the  ornithin  radicle.  The  correctness  of  this  supposition  has 
since  been  established  through  the  synthesis  of  arginin  from  cyana- 
mide  and  ornithin.  Ornithin,  moreover,  is  now  known  to  be  n, 
o-diamido- valerianic  acid,  and  the  structural .  formula  of  arginin 
may  hence  be  represented  as  follows  : 

I  I 

XH  =  C  —  NH.CH,.CH2.CH2.CH.COOH. 

Its  relation  to  guanidin  and  kreatin,  as  also  its  decomposition  into 
urea  and  ornithin,  is  further  shown  : 

cf(KH)  Cf(NH) 

\NH,  \N(CH3).CH2.C00H 

Guanidin.  Kreatin. 

/NH., 

CX.XH,  -  CH(CH,).NH2.C00H  =  CV-(XH) 
Cyanamide.  Mcthyl-glycocoll.  \N(CH3).CH2.C00H 

Kreatin. 

CX.XH,  ^  CH2.CH,.CH2.CH.COOH  =  CV(NH) 

I  I  \NH.CTLi.CH2.CH2.CH.COOH 

Cyanamide.     ^B.^  XH^  | 

Ornithin.  XH, 

Arginin. 
XH., 
Cf(XH) 
\XH.CH2.CH,.CIl2.CH.(XH2).COOH  -  H.O  = 
Arginin. 

.XH, 
C0(  -CH2(XH.,).CH2.CH,.CH(XTl2)COOH. 

\>sH.,  Ornithin. 

Urea. 

On  oxidizing  arginin  with  barium  permanganate  Kutscher 
obtained  guanidin,  guanidin-butyric  acid,  and  ethylene-succinic  acid. 
He  concludes  that  the  process  occurs  in  two  j)hases,  as  represented 
by  the  equations : 

82 


THE  HEXON  BASES.  83 

(1)  C-NH  I  +20  = 

\NH.  CHj.CH,.  CH,.  CH.  COOH 

Arginin. 

C^NH  +CO2  +  NH3 

\nH.  CH2.CH2.CH2.COOH 

Guauidin-butyricacid. 

(2)  C:  •  NH  +  20  = 

XNH.CHj.CH^.CHo.COOH 

Guanidin-butyric  acid. 

C^.NH  +COOH.CH2.CH2.COOH 

\NH,  Succinic  acid. 

Guanidin. 

Of  great  interest,  further,  is  the  fact  that  ornitliin  can  give  rise 
to  putrescin,  viz.,  to  tetramethylene-diaraine,  a  ptomain  which  is 
formed  during  the  putrefaction  of  albuminous  material,  and  which 
has  also  been  found  in  the  urine  in  association  with  cystin.  Thus 
far  this  transformation  has  been  effected  only  through  the  agency  of 
micro-organisms,  but  there  is  no  reason  to  suppose  that  their  presence 
is  essential,  and  that  in  the  tissues  of  the  living  body  the  same  proc- 
ess cannot  also  occur.  This  transformation  may  be  represented  by 
the  equation  : 

CH.,(NH,).CH,.CH._,.CH.(NH2).C00H  =  CO,  +  CH2(NH2).CH2.CH.,.CH2.(NH2) 
Ornithin.  Putrescin. 

Should  the  formula  of  ornithin,  as  above  indicated,  be  correct — 
and  it  may  be  added  that  there  is  every  ^reason  to  suppose  that  this 
is  the  case — we  can  readily  understand  how  'pyridin  derivatives  can 
develop  from  the  albumins  without  being  forced  to  assume  the 
existence  of  a  pyridin  radicle  in  the  albuminous  molecule  directly. 
The  active  principle  of  the  suprarenal  gland,  which  von  Fiirth 
regards  as  tetrahydro-dioxyiyyridin,  could  thus  result  from  ornithin 
by  the  replacement  of  the  «-amido-group  by  liydroxyl  and  the  elim- 
ination of  water.  That  oxypiperidin  results  from  o-amido-valerianic 
acid  in  an  analogous  manner  has  indeed  been  demonstrated.  These 
relations  may  be  expressed  by  the  formulse : 

CH2(NH.,).CH.,.CH,.CII.,.C00H  =  H.,0  +  CH.,(XH).CH.,.CH2.CH2.CO. 
5-amido-valerianic  acid.  oxypiperidin. 

(1)  CH.,(NH,).CH,.CH,.CH(NH.,).COOH  +  H,0  = 

Ornithin:  CH2(NH2VCH.,.CHj.CHfOH).COOH  +  NH3. 

a-liydroxy,  Samido- valerianic  acid. 

(2)  CH./NH2).CH,.CH2.CH(OH).COOH  ^ 

H,0  +  CHjfNH).CH2.CH,,.CHfOH).CO 
Tetrahydro-dioxy  pyridin. 

Lysin. — Lysin  is  apparently  a  homologue  of  ornithin,  and  is 
represented  by  the  formula  CH2(XH2).CH..CH..CH2.CH(NH2). 
COOH ;    it    is    thus   «,  £-diamido-capronic    acid.     On    hydrolytic 


84      THE  yiTROGEXOrS  DERIVATIVES  OF  THE  ALBUMIXS. 

deooinposition  it  yields  ammonia,  oxalic  acid,  propionic  acid,  and 
notably  acetic  acid.  On  oxidation  of  lysin  with  barium  perman- 
ganate Zickgraf  obtained  hvdrocvanic  acid,  oxalic  acid,  normal 
glutaric  acid  (COOH.CH,.CH,.CH,.COOH),  and  probably  also 
glutaniinic  acid.  When  exposed  to  the  influence  of  putrefactive 
organisms  it  gives  rise  to  the  formation  of  cadaverin — penta-methy- 
lene-diamin — a  ptomain  which  is  frequently  found  together  with 
putrescin  in  putrefying  albuminous  material,  and,  like  this,  may 
also  appear  in  the  urine  in  association  with  cystin.  Its  formation 
is  quite  analogous  to  that  of  putrescin  from  ornithin,  and  may  be 
represented  by  the  equation  : 

CIL,(NH,).CH2.CH,.CHj.CH(XH2).COOH  = 

Lysin.  CO,  -  CHj  ( yn^  I  .CH,.CH,.  CH2.CH.,  ( XHj ) 

Cadaverin. 

In  this  manner,  also,  albumins  can  give  rise  to  the  formation  of 
pyridins,  and,  as  a  matter  of  fact,  piperidin  results  during  the  dry 
distillation  of  cadaverin,  as  represented  by  the  ec^uation  : 

CH2-CH, 

CHjCNH^  .CH2.CH2.CH2.CH2!yH2J  =  CH^  ^'H  -  NHj. 

CH— CH, 

On  treating  lysin  with  benzoyl  chloride  Drechsel  obtained  a 
body,  of  the  formula  CgHi.lCOCgHjjoX^O.,  which  he  termed  h/surie 
acid,  and  which  is  thus  homologous  with  the  dibenzoyl  derivative 
of  ornithin,  C^Hj^^i COCgHJoXoO,,  omit h uric  aciJ. 

An  inactive  Ivsin   has  been  obtained  svntlietically  from  ;'-cyan- 

CO  f'  IT  \ 

propyl-malonyl   ether  (XC.(CHo)3.CH<^,q-^^,-jjM,  over  «-oximido- 

o-c;i-an-valerianic  ethyl  ether  iyC.{CR:)yC<y^'^'    ^).    On  racemi- 

sation  this  was  shown  to  be  identical  with  the  active  lysin. 

Histidin. — Of  the  nature  of  histidin  comparatively  little  is 
known.  It  is  formed  in  very  small  amounts  only  during  the 
decomposition  of  the  all)umins  and  may  be  absent.  Its  formula, 
according  to  Kossel,  is  C-HoX.p..  Structurally  it  is  possibly 
amido-methyl-dihydro-pyrimidin  carbonic  acid : 

HX— C 

HC    c— cH,.xai 

II   II 

N— C— COOH 

According  to  Herzog,  it  gives  the  biuret  reaction,  at  first  a  violet 
color  which  srraduallv  changes  to  red. 

For  the  isolation"  of  arginin.  lysin,  and  histidin  the  reader  is 
referred  to  Kossel  and  Kutscher's  paper,  '"  Beitriige  z.  Kenntniss  d. 
EiweisskGriH'r,"  Zeit.f.  phys.  Chem.,  1^00,  vol.  xxxi.,  p.  165. 


THE  MONO-AMIDO-ACIDS.  85 

THE   MONO-AMIDO-ACIDS. 

The  raono-amido-acid  derivatives  of  the  albumins  are  glycocoll 
(glycin),  alanin,  aniido-valerianic  acid,  leucin,  asparaginic  acid, 
glutaminic  acid,  phenyl-alanin,  serin,  tyrosin,  a-pyrrolidin-carbonic 
acid,  oxypyrrolidinc-arbonic  acid,  tryptophan,  and  cystin.  Of  these, 
glycocoll,  alanin,  amido-valerianic  acid,  and  leucin  are  amido 
derivatives  of  monobasic  acids  of  the  formic  series. 

Glycocoll  is  amido-acetic  acid  :  CH2(NH2).COOH. 

Alanin  is  «-amido-propionic  acid  :  CH3.CH(NH2).COOH. 

Leucin  is  a-amido-isobutyl-acetic  acid  :  (CH.3).,.CH.CH2.CH- 
(NH.).COOH. 

Asparaginic  acid  and  glutaminic  acid  are  dibasic  acids  of  the 
oxalic  series  : 

Asparaginic  acid  isamido-succinic  acid  :  CH2.CH(NH2).(COOH)2. 

Glutaminicacid  isamido-glutaricacid  :(CH2)2.CH(NH2).(COOH)J. 

Phenyl-alanin  is  a  representative  of  the  non-hydroxylated  acids  of 
the  aromatic  series,  viz.,  phenyl  a-amido-propionic  acid  :  CgHg.CHg.- 
CH(NH2).COOH. 

Tyrosin  is  the  corresponding  oxy-acid  ;  it  is  oxyj)henyl-alanin  or 
para-ox  vphenyl  a-amido-propionic  acid  :  C6H^(OH).CH2.CH(NH.,).- 
COOH(l  :  4). 

«-Pyrrolidin-carbonic  acid  is  a  pyrrol  derivative  :  CjH7(NH2)- 
COOH ;  and  oxypvrrolidin-carbonic  acid  the  corresponding  oxy-acid : 
C\H,(OH)(NH2).COOH. 

Serin  is  an  amido  derivative  of  glvcerinic  (propandiolic)  acid : 
C2H3(OH).(NH2).COOH. 

Tryptophan  is  a  skatol  derivative,  viz.,  skatol-amido-acetic  acid  : 
C„H,2N202. 

Cystin  is  an  amido-sulphur  compound  ;  it  is  the  disulphide  of  p'-cys- 
tein,  a-amido-/9-thiolactic  acid — i.  e.,  a-diamido-/3-dithio-dilactic 
acid. 

All  the  amido-acids  mentioned  are  albuminous  derivatives  and 
represent  integral  constituents  of  the  albuminous  molecule.  Their 
quantitative  relations,  as  has  been  indicated,  are  not  constant,  how- 
ever, and  upon  these  variations  no  doubt  the  characteristics  of  the 
individual  members  of  the  group  are  in  great  part  dependent  (see 
page  47). 

The  amido-acids  of  the  fatty  series  are  of  special  interest  to  the 
physiological  chemist,  owing  to  the  fact  that  they  are  a])parently 
intimately  concerned  in  the  jiroduction  of  urea.  Von  Schroder, 
Nencki,  and  others  have  thus  shown  that  in  the  liver  the  ammonium 
salt  of  carbamic  acid,  viz.,  amido-formic  acid,  is  transformed  into 
urea,  and  we  also  know  that  in  the  mammalian  organism  leucin, 
glycocoll,  and  asparaginic  acid  are  likewise  transformed  into  urea 
and  eliminated  as  such.  That  a  portion  of  the  urea,  indeed,  origin- 
ates in  this  manner  can  scarcely  be  doubted.  As  regards  the 
nature  of  the  chemical  changes  which  take  place  during  the  trans- 


86      THE  yTTROGESOUS  DERIVATIVES  OF  THE  ALBUMINS. 

formation  of  the  amido-aeids  into  nrea,  onr  knowledge  is .  not 
conii)lete.  It  was  formerly  supposed  that  uric  acid  represented  the 
immediate  antecedent  of  urea  and  was  transformed  into  this  by 
oxidation.  AVe  find,  as  a  matter  of  fact,  that  in  birds  and  reptiles 
uric  acid  constitutes  tlie  final  decomposition-product  of  the  nitro- 
(reuous  metabolism,  and  is  thus  analogous  to  the  urea  of  mammals. 
I  have  also  p(tinted  out  that  as  a  ureid,  uric  acid  on  oxidation 
can  yield  urea.  But  as  far  as  is  known,  the  uric  acid  of  mammals  is 
exclusively  derived  from  the  nucleinic  bases,  and  is  thus  scarcely 
formed  in  sufficient  quantity  to  give  rise  to  the  large  amount  of  urea 
which  is  daily  eliminated  in  the  urine.  That  a  small  fraction  of 
the  urea  may  result  from  uric  acid  by  simple  oxidation  is  possible, 
and  indeed  prol)able,  but  the  greater  portion  must  of  necessity 
originate  in  a  diti'erent  manner. 

In  birds,  on  the  other  hand,  some  of  the  uric  acid  apparently 
results  from  glycocoll,  and  we  thus  see  that  in  both  classes  of  animals 
the  amido-acids  may  be  the  antecedents  of  the  final  })roducts  of 
nitrogenous  metabolism. 

It  is  conceivable  that  in  mammals  cyanic  acid  may  be  produced 
as  an  intermediary  product,  and  that  urea  then  results  tlirough  a 
condensation  of  two  molecules  of  this  substance  in  datu,  nascendi, 
according  to  the  equation  : 

CONH  +  CONH  =  CCK  +  CO^. 

Then  again  we  may  imagine  that  a  transformation  of  the  amido- 
acids  occurs  into  the  ammonium  salts  of  the  fatty  acids  standing 
next  in  order  in  the  downward  scale,  and  that  by  further  oxidation 
these  are  transformed  into  ammoniiun  carbonate,  and  this  into  urea. 
ft  has  been  shown  as  a  matter  of  fact  that  a  fair  amount  of  urea  is 
tims  produced  when  blood  containing  ammonium  carbonate  or  ammo- 
nium formate  is  allowed  to  flow  through  the  isolated  livers  of  dogs. 
According  to  Drechsel,  finally,  the  amido-acids  are  transformed 
into  carbamic  acid,  fn)m  which  urea  may  then  result,  as  indicated  by 
the  equation  : 


/NiT2  ySH,  /NII2 

a)/       +  co<       =  co(        +  CO2  +  HjO. 

\0H  ^OH  ^NH, 


In  a  subsequent  chapter  this  subject  will  be  treated  at  greater  detail. 

In  addition  to  the  important  role,  which  the  amido-acids  thus 
play  in  the  formation  of  urea,  these  bodies  are  of  further  interest 
from  the  part  which  they  tak(!  in  some  of  the  syntheses  that 
occur  in  the  animal  organism.  In  this  manner  they  give  rise  to  a 
number  of  complex  substances  wliich  can  hence  be  regarded  as 
amido-derivatives.  With  benzoic  acid  glycocoll  thus  combines  to 
form  hi})[)uric  acid,  as  shown  by  the  equation  : 

CII,(NH,).COOH  +  CsHj.COOH  =  CH2.NII(CfiniCO).CO0H  -  H,0 
Glycocoll.  Benzoic  acid.  Hippuric  acid. 


THE  MONO- A MIDO- ACIDS.  87 

With  phenyl-acetic  acid  glycocoll  similarly  combines  to  form 
phenaceturic  acid  : 

CH2.(NH2).COOH  +  CH2.(C8H5).COOH  = 

CfiHj.CH^.CO  —  NH.CH,.CO0H  ^  R,0. 

That  uric  acid  on  iiydrolytic  decomposition  will  yield  ammonia, 
carbon  dioxide,  and  glycocoll  has  been  shown.  There  is  evidence, 
moreover,  to  show  that  in  tlie  organism  of  birds  and  reptiles,  at 
least,  its  synthesis  can  similarly  occur. 

Ornithuric  acid  results  through  the  union  of  benzoic  acid  with  the 
diamide  ornithin  ; 

C^HjlNHjjjj.COOH  H-  2C6H5.C(XJH  =  C,H,fNH.C«H5.C()),.(X>0H  +  2H,0 
Ornithin.  Benzoic  acid.  Ornithuric  acid. 

The  amido-acids  also  are  clo.sely  related  to  the  biliary  acids,  as  on 
decomposition  with  baryta-water,  glycocholic  acid  is  decomposed 
into  glycocoll  and  cholalic  acid,  as  shown  in  the  equation  : 

C^eH^NOe  +  ILp  =  CH,(NH.j.COOH  -f  C,,H«,05 
Glycoctiolic  Glycocoll.  Cholalic 

acid.  acid. 

Taurocholic  acid  similarly  gives  rise  to  cholalic  acid  and  taurin, 

which  latter  can  be  regarded  as   amido-isethionic  acid — that  is,  as 

isethionic  acid,  Yl{CJi^.OWpO■^,  \n  which  the  hydroxyl  group 
has  been  replaced  by  the  amido-radicle  : 

CV,H^NO,S  +  H-P  =  C^^H^Oj  +  C,H7^^03S 
Taurocholic  Cholalic  Taurin. 

acid.  acid. 

After  the  ingestion  of  amido-acids,  moreover,  the  corresponding 
compounds  of  carbamide,  — CONH,  appear  in  the  urine.  These  are 
spoken  of  as  the  uramic  acids,  and  compri.se  methyl-hydantoinic  acid, 
taurocarbamic  acid,  uramido-benzfjic  acid,  and  tyrosin-hydantoinic 
acid,  or  hydantoin-hydroparacumaric  acid.  They  are  found  after 
the  ingestion  of  sarcosin  or  methyl-glycocoll,  of  taurin,  amido- 
benzoic  acid,  and  tyrosin,  respectively.  The  syntheses  which  are 
thus  effected  may  be  represented  by  the  equations  : 

C,H.O.,      +  CONH  =  C,HhN.P, 

Sarcosin  Methyl-hydantoinic 

acid. 

aH-NS03+   CONH  =  C,H,N.,SO, 
Taurin  Taurocarbaunc 

acid. 

C,H,N02     +   CONH  =  C«H,N.P3 

Amidobenzoic  Uramido-benzoic 

acid.  acid. 

C,H„NO,   +  CONH  ^  C,„H,„NA   +   H.,0 
tyrosin.  Tvrosin-hydantoinic 

acid. 

The  cyst'in  complex  of  the  albuminous  molecule  is  the  mother- 
substance  of  taurin.     On  oxidation  it  is  first  transformed  into  cy.s- 


88      THE  NITROGENOUS  DERIVATIVES  OF  THE  ALBUMINS. 

teinic,  acid  and  by  loss  of  COo  this  gives   rise  to  taurin,  as  repre- 
sented by  the  equations : 

(1)  C6H,2NAS2  +  50  +  H,0  =  2C3H-NO5S 

Cystin.  Cysteiuic  acid. 

(2)  CaH^NOjS  —  CO2  =  C^H^NOaS 
Cy>tcinic  acid.  Taurin. 

Analogous  to  the  formation  of  taurocarbarainic  acid  from  taurin 
cystin  gives  rise  to  the  formation  of  cystin  hydantoinic  acid  on  treat- 
ing with  cyanic  acid  : 

On  reduction  cystin  yields  cystein : 

CH2.S— S.CH2  CH2.SH 

III 
CH.NH2     CH.NH2  +  2H  =  2CH.NH2 

i  I  I 

COOH        COOH  COOH 

Cystin.  Cystein. 

On  reduction  the  amido-acids  are  transformed  into  the  correspond- 
ing acids  from  ^vhich  they  are  derived.  Glycocoll  is  thus  transformed 
into  acetic  acid,  leucin  into  capronic  acid,  asparaginic  acid  into  suc- 
cinic acid,  glutaminic  acid  into  glutaric  acid,  tyrosin  into  para-oxy- 
phenyl-propionic  acid  (hydroparacumaric  acid),  etc.,  as  sho^vn  by 
the  equations : 

CH2(NH,,).C00H  +  2H  =  CH3.COOH  +  NHa 

Glycocoll.  Acetic  acid. 

/COOH  /COOH 

CH,.CH(NH„).<  +  2H  =  CH^-CH^./  +  NH3 

\COOH  \COOH 

Asparaginic  acid.  Succinic  acid. 

/OH  /OH 

CeH  /  +  2H  =  CgH  /  +  NH3 

\CH2.CH(NH2).COOH  ^CH^.CH^.COOH 

Tyrosin.  Para-oxy-phenyl-propionic 

acid. 

On  oxidation  these  are  further  changed  into  the  acids  standing 
next  in  order  in  the  downward  scale.  Acetic  acid  thus  gives  rise  to 
the  formation  of  formic  acid,  succinic  acid  to  malonic  acid,  para-oxy- 
phenyl-propionic  acid  to  para-oxy-phenyl-acetic  acid,  etc.,  as  shown 
by  the  equations : 

CH3.COOH  ^  30  =  H.COOH  +  H2O  +  COj 

Acetic  acid.  Formic  acid. 

/COOH  /COOH 

CH2.CH2./  +  30  =  CB./  +  HjO  +  COj 

^COOH  "^COOH 

Succinic  acid.  Malonic  acid. 

.OH  /OH 

C6H,<  +  30  =  CeH,  ^  +  H,0  +  CO, 

CH^.CH^.COOH  CH^.COOH 

Para-oxy-phenyl-propionic  Para-oxy-phenyl-acetic 

acid.  acid. " 

Through  a  splitting  oif  of  carbon  dioxide  para-oxy-])henyl-acetic 


THE  MONO- A MIDO- ACIDS.  89 

acid  then   further  gives  rise  to  paraeresol,  from   which  ])lienol  is 
finally  obtained  on  oxidation  : 

/OH  X)H 

CeH  /  =  QH,(         +  CO, 

^CH^-COOH  'CH, 

Paracresol. 

OH 
CgH/         +  30  --  CfiHs.OH  +  CO2  +  HjO 
^CHj  Phenol. 

In  the  animal  body  paracresol  and  phenol,  which  are  formed  from 
tyrosin  during  the  process  of  intestinal  putrefaction,  then  combine 
with  sulphuric  acid,  and  are  eliminated  through  the  urine  in  the 
form  of  their  potassium  salts  : 

/OH  /OH  /O.HSO3 

CfiH/         +S0/        ^CfiH/  +HA 

^CHg  \0H  \CH3 

/OH 
CfiH^OH      +  S02<         =  CgHj.O.HSO,     -i-  H,0. 
\0H 

Through  loss  of  CO.  tyrosin  yields  para-oxyphenyl-ethylamin  i 

C6H,(OH).CH.,.CH(NH2).COOH  =  CO,  +  C6H,(OH).CH,,.CH2.XH2 
Tyrosin.  Oxyphenyl-ethylamin. 

In  a  similar  manner  phenyl-alanin  can  give  rise  to  phenyl-ethy- 

1am in  : 

C6H5.CH.,.CH.NH2.COOH  =  CO,  +  CsHs.CH^.CH.NH, 
Phenyl-alanin.  Phenyl-elhylamin. 

The  onlv  indol  derivative  which  is  formed  from  the  albumins  on 
hvdrolysis  by  means  of  the  common  digestive  ferments  is  tryptophan 
(skatol-amido-acetic  acid) : 

/C(CH3)^ 
C«h/  ^C.CH(NH2).C00H 

\NH'^'^ 

This  can  now  be  regarded  as  themother-substanceof  the  aromatic 
products  indol,  skatol,  and  skatol-carbonic  acid,  which  are  con- 
stantly formed  from  the  albumins  by  putrefactive  oriianism.s.  In 
contradistinction  to  tyrosin  and  its  derivatives  these  bodies  belong 
to  the  ortho  series. 

Indol,  skatol,  and  skatol-carbonic  acid  are  closely  related  to  each 
other  and  to  indigo.  Skatol  thus  results  from  indol  through  a  sub- 
stitution of  the  methyl-group  for  a  hydrogen  atom  of  one  of  the  CH 
groups,  as  shown  by  the  formula  : 


/CH  ^  /C(CH3 

/        >CH  CgH,/       • 


Indol.  Skatol. 


90      THE  XITROGESOUS  DERIVATIVES  OF  THE  ALBUMINS. 

Skatol-carbonic  acid  then  results  from  skatol  through  a  union  with 
carbon  dioxide  : 

CfiH/  3C-C00H 

Skatol-carbonic  acid. 

In  their  passage  through  the  animal  body  indol  and  skatol  are 
oxidized  to  indoxyl  and  skatoxyl,  and  are  eliminated  in  the  urine  to 
a  great  extent,  in  combination  with  sulphuric  acid,  as  potassium 
indoxvl  sulphate  (animal  indican)  and  potassium  skatoxyl  sulphate, 
while  the  skatol-carbonic  acid  is  excreted  as  such.  These  changes 
can  be  expressed  by  the  equations  : 

(DCeH/        >CH-fO        =CeH/  J^CH 

Indol.  Indoxyl. 

/C(OH)  ^  ^C(0.S03H)^ 

"^  XH.^-^  ^  XH  ^^ 

Indoxyl.  Indican. 

On  decomposition  with  strong  hydrochloric  acid  indican  is  accord- 
ingly decomposed  into  sulphuric  acid  and  indoxyl,  which  latter  can 
then  be  oxidized  to  indigo-blue  : 

2C6H/  3cH  +  20  =  C6H/         >C  =  C<  >C6H, +  2H.,0 

\XH^^  \XH/  \XH/ 

Indoxyl.  Indigo-blue. 

On  reduction  indigo-blue  is  transformed  into  indigo-white, 
C^HgXO,  which,  when  boiled  with  zinc  and  water,  then  further 
yields  indol : 

CeH^XO  +  H    ^^CgHeXO. 

CgHfiXO  +  3H  :--  CgH-X  +  H.,0. 

Animal  indican,  however,  must  not  be  confused  with  vegetable 
indican,  which  is  a  glucoside,  and  yields  indigo-blue  and  indiglucin 
on  hydrolytic  decomposition : 

C26H31XO17  +  2H,0  =  C,H,XO  -^  SCeH.oOe 
Vegetable  indican.  Indigo-blue.      Indiglucin. 

A  small  amount  of  skatoxyl,  indoxyl,  and  phenol  is  also  eliminated 
in  the  urine,  in  combination  with  glucuronic  acid,  as  skatoxyl, 
indoxyl,  and  ])henol  glucuronates,  respectively.  This  acid  may  be 
derived  from  glucose  by  the  substitution  of  one  atom  of  oxygen  for 
two  atoms  of  hydrogen,  and  is  accordingly  represented  bv  the 
fornmla  COOH.(CH.OH),.COH.  On  oxidation  it  is  transformed 
into  .saccharinic  acid,  the  relation  of  whicii  to  gluco.se  has  already 
been  considered. 


THE  ORGANIC  NOX-NITBOGENOUS  ACIDS.  91 


THE   ORGANIC   NON-NITROGENOUS   ACIDS. 

The  organic  non-nitrogenous  acids  which  are  formed  in  the  animal 
body  are  largely  members  of  the  fatty  acid  series.  Others  belong  to 
the  glycolic  series,  still  otiiers  to  the  acrylic  series,  some  are  repre- 
sentatives of  the  oxalic  series,  and  still  others  belong  to  the  aromatic 
oxy-acids.  The  first  group  comprises  the  following  acids,  which  as 
a  class  may  be  represented  by  the  formula  C„H2„02. 

Formic  acid HCOOH  =  CHA 

Acetic  acid CH3.COOH  =  CH.O^ 

Propionic  acid CH,.CH,.COOH        =  CaHgO^ 

Butyric  acid (CHj)2.CH3.COOH   =  C^HgO,, 

Valerianic  acid  ....  (CH2)3.CH3.COOH   =  C^HjoOj 

Capronicacid (CH^i.-CHs-COOH   =  CgHiA 

Palmitic  acid (CH.^li^.CHj.COOH  =  C.sHgA 

Stearic  acid (CH2),6.CH3.COOH  =  C^HjeO^ 

The  two  last,  as  has  been  seen,  are  integral  constituents  of  the 
fats,  in  which  they  are  present  in  combination  with  glycerin  as  tri- 
glycerides. From  these  the  others  may  in  part  be  derived,  but  to 
the  greatest  extent  no  doubt  they  result  from  the  amido-acids 
through  a  process  of  oxidation  or  reduction,  as  illustrated  by  the 
equations : 

CH2(NH,).C00H  +  20  =  H.COOH  +  NH3  +  COj 
GlycocoU.  Formic 

acid. 

CH.,(>'H,).COOH  +  2H  =  CH,.COOH  +  JsHs 

GlycocoU.  Acetic  acid. 

Through  further  oxidation  these  acids  are  then  transformed  into 
those  standing  next  in  order  in  the  downward  scale,  and  so  on, 
until  finally  carbon  dioxide  and  water  re.sult,  as  seen  in  the  equations  : 

(1)  CH2.CH3.COOH  +  30  =  CH3.COOH  +  CO2  +  H2O 

Propionic  acid.  Acetic  acid. 

(2)  CH3.COOH  +30  =  H.COOH      +C02  +  HjO 

Acetic  acid.  Formic 

acid. 

(3)  H.COOH  +  O    =  CO2  +  H2O 
Formic  acid. 

To  some  extent,  however,  the  fatty  acids  are  derived  also  from  lactic 
acid  and  related  acids,  which,  as  will  be  seen  later,  are  constantly 
being  formed  in  the  metabolism  of  the  albumins.  Their  transforma- 
tion into  the  fatty  acids  is  then  probably  analogous  to  the  production 
of  butyric  acid  during  the  process  of  butyric  acid  fermentation. 

2C3HSO3  =  C,H«0.,  ^  2CO2  -  4H 

Lactic  Butyric 

acid.  acid. 

The  glycolic  acids  which  are  found  in  the  animal  body  may  be 
represented  by  the  general  formula  CJi^nO^.  They  comprise  the 
following;  bodies : 


92      THE  yiTROGESOUS  DERIVATIVES  OF  THE  ALBUMINS. 

Glvcolicacid CH.,.OH.COOH  =;  CH.Og 

Ethyliclene-lactlcacid(paral:icticacid)   .    CII.j.CH(()H).COOH  =  CiligOs 

Ethvlidene  lactic acid(opticallv  inactive )  CHij.CHi UH).COOH  =  CtHfiOj 

Etlivlidene-lffivo-lacticacid  .."....    CH3  CH(OH).COOH  =C'n,.03 

/3-oxvbutvric  acid CH.0H.CH,.CH3.C00H  =  C.'H^Og 

Leucinicacid (CH3)2.CH.CH2.CH.OH.COOH  =  CeH.jOj 

Of  these  aciils,  glycolic  acid  does  not  occur  in  the  animal  body,  so 
far  as  is  known,  but  it  is  of  interest  owing  to  the  fact  that  it  is 
closely  related  to  glycocoll,  and  is  derived  from  this  in  the  same 
manner  in  which  leucinic  acid  is  obtained  from  leucin,  viz.,  by  the 
substitution  of  a  hydroxyl  group  for  the  amido-group,  as  shown  by 
the  equations : 

CH2.(NH2).COOH     +  H.p  =  CH,(OH).COOH  +NH3 
Glycocoll.  Glycolie  acid. 

(CH3).,.CH.CH,,.CH.(NH2).COOH  +  H^O  = 

Leuciu.  (CH3)2.CH.CH2.CH(OH).COOH  +  NH3 

Leucinic  acid. 

;9-oxybutyric  acid  is  found  only  under  pathologic  conditions. 

Lactic  acid  and  its  isomeric  compounds,  as  well  as  leucinic  acid 
and  ;5-oxy butyric  acid,  are  all  albuminous  decomposition-products, 
and  in  part,  at  least,  derived  from  the  amido-acids.  The  origin  of 
lactic  acid,  however,  is  not  so  clear,  but  we  shall  consider  this  ques- 
tion in  greater  detail  in  a  subsequent  chapter. 

On  reduction  these  acids  can  be  transformed  into  fatty  acids. 
Lactic  acid,  as  has  just  been  shown,  thus  gives  rise  to  butyric  acid, 
leucinic  acid  is  similarly  changed  to  capronic  acid,  etc. 

On  oxidation  /9-oxybntyric  acid  is  transformed  into  diacetic  acid, 
which  in  turn  is  decomposed,  Avith  the  formation  of  acetone  and 
carbon  dioxide : 

CH3.CH.0H.CH.,.C00H  +  O  =  (CH3.C0)CH.,  C'OOH  +  H^O 
^-oxybutyric  acid.  Diacetic  acid. 

(CH3.C0)CH.,.C00H  =  C0(CH,)2  +  CO., 
Acetone. 

The  bodies  which  are  thus  formed  are  of  sjiecial  interest  to  the 
pathologist,  as  their  accumulation  in  the  animal  body  is  apparently 
capable  of  causing  very  serious  disturbances.  Acetone,  however,  is 
also  met  with  under  normal  conditions,  and  apparently  represents  a 
constant  ]iroduct  of  albuminous  decomposition. 

On  boiling  with  dilute  mineral  acids  /5-oxybutyric  acid  is  trans- 
formed into  an  acid  of  the  acrylic  series  (see  below),  viz.,  a-crotouic 
acid  : 

CH3.CH(OH).CH2.COOH  =  2H2O  +  CH3  —  CH  =  CH.COOH 

o-Crotonic  acid. 

The  acids  of  the  acrylic  series  can  be  represented  by  the  general 
formula  CnH^n.^O^.  Representatives  of  these  are  the  «-capronic 
acid,  just  referred  to,  and  oleic  acid,  M'hich  as  a  triglyceride  rep- 
resents a  most  important  constituent  of  many  of  the  vegetable  and 
animal  fats.    On  heating  with  hydriodic  acid  and  red  phos[)horus  to 


THE  ORGANIC  NON-NITROGENOUS  ACIDS.  93 

a  temperature  of  210°  C,  oleic  acid  takes  up  two  atoms  of  hydro- 
gen, and  is  thus  reduced  to  stearic  acid  : 

CH3.(CH,),3.CH  =  CH.CH2.COOH  +  2H  =  CH3.(CH,),e.C00H 
Oleic  acid.  Stearic  acid. 

On  treating  with  nitrous  acid,  in  statu  nascendi,  it  is  transformed 
into  the  solid  elaidinic  acid,  which  is  isomeric  with  oleic  acid  and 
belongs  to  the  same  series. 

The  dibasic  acids  of  the  oxalic  series  can  be  represented  by  the 
formula  C„Ho,j_20^.  They  include  oxalic  acid,  succinic  acid,  and 
glutaric  acid,  of  which  the  two  latter  are  generally  met  with  in  the 
form  of  their  amides,  viz.,  as  asparaginic  acid  and  glutaminic  acid, 
respectively.  In  this  form  they  are  invariably  obtained,  together 
with  oxalic  acid,  as  decomposition-products  of  most  albumins. 
Oxalic  acid  and  succinic  acid,  however,  may  also  be  observed  as 
such. 

The  relation  of  oxalic  acid  to  the  ureids  has  already  been  con- 
sidered.    They  are  represented  by  the  formulae : 

Oxalic  acid (COOH),  =  C,,HA 

Succinic  acid      {CH2)2.(COOH)2 -^  C,Hb04 

Glutaric  acid (CHjtg.iCOOHj^  =  C5HP4 

On  oxidation  they  are  decomposed  into  water  and  carbon  dioxide, 
but  it  is  probable  that  during  this  transformation  fatty  acids  are 
formed  as  intermediary  products : 

CJifii  =  CO2  +  H.COOH. 

C.HeO,  =  CO2  +  C,H«0, 

Propionic 
acid. 

The  principal  aromatic  oxy-acids   which   may  be   found   in   the 

animal  body  are  hydroparacumaric  acid    or  para-oxy-])henyl-pro- 

pionic    acid,    para-oxy-phcnyl-acetic     acid,    para-oxy-phenyl-lactic 

acid  or  oxy-hydroparacumaric  acid,  and    para-oxy-phenyl-glycolic 

acid.     They  are  derivatives  of  phenol,  in  which  one  hydrogen  atom 

has  been  replaced  by  the  radicle  of  the  corresponding  non-aromatic 

acid,  as  shown  by  the  equation  : 

/OH 
CfiH^.OH  +  CH3.CH.OH  COOH  +  O  =  CeH^  +  H^O 

Phenol.  Sarcolactic  acid.  ^CHj.  CH  OH.  COOH 

Para-oxy-phenyl- 
lactic  acid. 

They  are  probably  all  derivatives  of  tyrosin,  and  it  has  already 
been  shown  (p.  89)  how  hydroparacumaric  acid  results  from  this  on 
reduction,  and  is  then  transformed  into  para-oxy-phenyl-acetic  acid 
by  oxidation. 

The  formulae  of  these  acids  and  their  relation  to  tyrosin  are  as 
follows : 


94      THE  NITROGENOUS  DERIVATIVES  OF  THE  ALBUMINS. 

Tyrosin  (para-oxy-phenvl-amido-propionic  acid)  =  C6H4<(^ 

\CH2.CH(NH2).COOH 

/OH 
Para-oxv-phenvl-propionic  acid  =  C6H^<^ 

\CH2.CH2.COOH 

/OH 

Para-oxy-plienyl-acetic  acid        =  C^i'C 

\CH2.COOH 

/OH 
Para-ox y-plienyl-lactic  acid         =  CgH^x 

\CH,.CH.OH.COOH 


/OH 
Para-oxv-olienyl-glycolic  acid     =  CgH^^^ 

^CH.OHCOOH 


On  decomposition  these  acids  yield  phenol  or  cresol,  water,  and 
carbon  dioxide,  as  shown  by  the  equations  : 

/OH  /OH 

(1)  QH  /  +  30  =  QH  /  +  CO,  +  H.p. 

\CH2.CH2.COOH  \CH2.COOH 

/OH  /OH 

(2j  QH  /  +  30  =  CgH  /  +  CO,  +  HP 

^CH^.COOH  \COOH 

Para-oxy- 
benzoic  acid. 

/OH 
(3)  CfiH  /  =  CeH^.OH  +  CO, 

^COOH  Phenol. 

or 

/OH  /OH 

(1)  QH/  =C6H/         +CO3 

\CH,.COOH  \CH3 

Paracresol. 

OH 

(2)  CeH,<         +  30     =  QH5.OH  +  CO,  +  H,0 

\CH3  Phenol. 

From  phenol  the  two  dioxybenzols  jjyrocatechin  and  hydro- 
qninon  can  then  result,  and  appear  in  the  urine  together  wi^^h 
phenol  as  conjugate  sulphates  or  glucuronates. 

.0H(1) 
CeH,.OH  +  O  =  CeH,.; 

\0H(2)  or  (4). 

Of  the  non-hydroxylated  aromatic  acids,  two  are  found  in  the 
animal  body,  and,  like  the  hydroxylated  compounds,  originate  during 
the  process  of  albuminous  putrefaction.  These  are  phenyl-propionic 
acid  (hydrocinnamic  acid)  and  phenyl-acetic  acid.  They  are  rep- 
resented by  the  formuke  : 

C6H5.CH.,.CH.,.COOH  =  phenyl-propionic  acid 
CgHs.CH,  COOH  —  phenyl-acetic  acid. 


THE  NUCLEINIC  ACIDS.  95 

By  combining  with  glycocoll  they  give  rise  to  the  formation  of 
hippuric  acid  and  phenaceturic  acid,  respectively.  In  the  first 
instance,  however,  benzoic  acid  is  apparently  first  formed,  which 
then  unites  Avith  glycocoll,  as  shown  in  the  equations  below : 

(1)  C6H5.CH,.CH2.COOH  +  60  =  CeHs.COOH  +  2CO2  +  2H2O 

Phenyl-propionic  Benzoic  acid, 

acid. 

(2)  CgHj.COOH  +  CH2(NH2).COOH        =:  C6H-.C0.CH.,.NH.C00H  +  H^O 
Benzoic  acid.  Glycocoll.  Hippuric  acid. 

and 

C6H5.CH,.COOH  +  CH2(NH2).COOH  =  CeHj.CH^.CO.NH.CHs.COOH  +  H^O 
Phenyl-acetic  Phenaceturic  acid, 

acid. 

The  phenyl-acetic  acid  is  no  doubt  derived  from  phenyl-alanin. 
just  as  para-oxyphenyl  acetic  acid  results  from  tyrosin. 

(1)  C6H5.CH2.CH.NH2.COOH  +  2H  =  C6H5.CH2.CH2.COOH  +  NH3 

Phenyl-alanin.  Phenyl-propionic  acid. 

(2)  C6H5.CH2.CH.,.COOH  +  30  =  CrHj. CH^. COOH  -f  CO^  +  H^O 
Phenyl-propionic  acid.  Phenyl-acetic  acid. 


THE  NUCLEINIC  ACIDS. 

The  nucleinic  acids  are  specific  decomposition-products  of  the 
nucleins  (viz.,  nucleoproteids),  and  are  characterized  by  the  fact 
that  on  hydrolysis  with  certain  mineral  acids  they  yield  phosphoric 
acid,  purin  bases,  and  frequently  also  a  pyrimidin  derivative, 
thymin.  All  nucleinic  acids  also  contain  at  least  one  carbohydrate 
group. 

Diiferent  nucleinic  acids  exist,  which  show  a  somewhat  different 
composition  according  to  the  nucleoproteids  from  which  thev  are 
derived.  According  to  their  origin,  they  are  termed  :  spermato- 
nucleinic  acid,  thyraonucleinic  acid,  triticonucleinic  acid,  yeast 
nucleinic  acid,  etc.  Such  as  they  are  obtained  from  the  tissues  the 
nucleinic  acids  possibly  represent  mixtures  of  different  nucleinic 
acids  of  a  simpler  order.  Neumann  has  pointed  out  that  on  treating 
the  nucleoproteid  of  the  thymus,  spleen,  pancreas,  and  the  testicles 
of  the  ox,  with  caustic  alkali,  a  nucleiniG  acid-a  results,  which  is 
characterized  by  the  fact  that  it  gelatinizes  in  5  per  cent,  or  in 
stronger  solution.  This  nucleinic  acid-a  apparently  represents  the 
native  acid.  On  further  treatment  with  caustic  alkali  it  passes  over 
into  the  nucleinic  acid-j3  through  a  process  of  depolymerizatjon. 
This  second  acid  does  not  gelatinize,  and  is  more  readily  soluble 
than  the  a  form.  During  this  transformation  two-thirds  of  the 
nucleinic  bases  are  split  off.  Elementary  analysis  of  the  two  forms 
(obtained  from  the  thymus)  has  given  the  following  results  : 

P-acid CgoHi^jOeiNj.Pio 


S6      THE  yiTROGESOUS  DERIVATIVES  OF  THE  ALBUMINS. 

Kossel  has  expressed  the  opinion  tliat  in  reality  only  four  true 
nuclcinic  acids  exist,  in  each  of  which  only  one  nucleinic  base  is 
represented.  He  accordingly  distinsruishes  an  adenylic  acid,  a 
guanylic  acid,  a  sarcylic  (hypoxanthylicj  acid,  and  a  xanthylic  acid. 
In  accordance  with  this  supposition,  the  spermatonucleinic  acid  of 
the  ox  would  contain  three  primary  acids,  as  on  decom])osition  it 
yields  xanthin,  hypoxanthin,  and  adenin.  Triticonucleinic  acid 
would  similarly  represent  a  mixture  of  guanylic  acid  and  adenylic 
acid  ;  thvmonucleinic  acid  likewise  contains  both  guanin  and  adenin, 
etc.  As  a  matter  of  fact,  however,  only  one  nucleinic  acid  has  as 
vet  been  isolated  in  pure  form,  which  actually  contains  but  one 
nucleinic  base  -,  the  rest  are  hypothetical.  The  one  acid  is  Bang's 
guanylic  acid,  which  was  obtained  from  the  pancreas.  Kossel's 
adenylic  acid,  which  was  first  regarded  as  an  analogous  product,  was 
shown  to  yield  both  guanin  and  adenin. 

As  a  class  the  nucleinic  acids  are  but  little  soluble  in  cold  water, 
more  readily  so  in  hot  water,  and  easily  soluble  in  solutions  of  the 
alkalies.  They  have  not  yet  been  obtained  in  crystalline  form. 
They  are  dibasic  acids,  and  form  both  acid  and  neutral  salts  with  the 
alkalies  and  the  heavy  metals.  They  are  precipitated  by  the  mineral 
acids,  but  not  by  acetic  acid,  with  the  exception  of  guanylic  acid.  They 
are  very  readily  decomposed  on  boiling  with  mineral  acids  and 
even  with  water,  while  they  are  quite  resistent  to  alkalies.  Alcohol, 
especially  acid  alcohol,  causes  their  precipitation  ;  ammonium  sul- 
phate in  the  presence  of  acetic  acid  is  equally  effective.  Tannic  acid, 
picric  acid,  and  phosphotungstic  acid  may  also  be  employed. 

All  nucleinic  acids  give  the  reaction  of  Adamkiewicz,  the  xantho- 
proteic reaction,  and  a  marked  reaction  with  phloroglucin  and  hydro- 
chloric acid  (see  Pentoses). 

In  acid  solutions  they  form  precipitates  with  albumins  which, 
according  to  Kossel  and  Milroy,  closely  resemble  the  native 
nucleins.  In  the  spermatozoa  of  fish  nucleinic  acid  is  present  as  a 
neutral  or  acid  salt  of  protamin.  The  elementary  composition  of 
the  common  forms  is  given  below  : 

(4uanvlic  acid C4,H66^^oP403^  (Bang) 

Triticonucleinic  acid       .    .    .  Q,PIfiiN,6PAi  (Osborne  and  Harris) 

Yeast  nucleinic  acid   ....  C4oH6oX]6P40,2  (Miescher) 

Salmonucleinic  acid C4oH56Ni4P40m  (Schmiedeberg) 

Thvmonucleinic  acid  .    .    .    .  Co^HjeN,  PAo  (Kossel) 

Ino.'^inic  acid.    .        CioHuN^  P  Og  (Liebig  and  Haiser) 

The  structural  composition  of  the  nucleinic  acids  is  still  largely 
mh  jndice.  For  triticonucleinic  acid  Osborne  has  suggested  the 
following  formula,  in  which  x  represents  an  as  yet  unresolved 
residue : 


THE  NUCLEINIC  ACIDS.  97 

/OH 

qha-pCqha 

I  ^OH 
O 

OH-.P-C,H3N202 

X       I 
O 

I 
OH— P-OH 

r^C^HgNp, 

cyi.N.o-^  I 

OH 


For  guanylic  acid  Bang  has  suggested  the  structural  formula 


OH^^OH  OH 

C,H,N,-0-P-0    C3H5\,__ 


5^  ^9^5 


0 

1 

/OH 

CaH.Ns- 

-0- 

-P— 0- 

-C3H 

'^C,HA 

/ 

''  \ 

0 

o 

^.  OH 

CsH.N^- 

-0- 

-P-0- 

-C3H 

'"^C^HgOs 

1 

0 

C,H,N5- 

-0- 

-P— OH 

/\ 

HO 

OH 

Some  niicleinic  acids,  such  as  thymoniicleinic  acid,  contain  iron 
in  masked  form. 

As  I  have  already  indicated,  the  native  nucleinic  acids  on  hydro- 
lysis lose  a  portion  of  their  xanthin  bases  and  give  rise  to  the  for- 
mation of /9-nucleinic  acid.  On  further  decomposition  the  remain- 
ing xanthin-bases  are  split  off  together  with  another  basic  substance 
which  has  been  termed  cytosin.  A  body  then  remains  which  con- 
tains the  residual  radicles  of  the  nucleinic  acid  molecule.  This 
Kossel  and  Neumann  have  termed  thyminic  acid.  For  its  barium 
salt  they  obtained  the  formula  Ci4H2.,]V30,2P2Ba,  and  in  the  ease  of 
the  thyminic  acid  resulting  from  triticonucleinic  acid  Osborne  has 
suggested  the  structural  formula  : 


98      THE  yiTROGESOVS  DERIVATIVES   OF  THE  ALBCMISS. 

OH 
C\H,0,-P— C,H,0, 


l\0H 


OH 


>P-C,H3N,0, 

O 

1  /OH 
OH— P( 

1  ^QHsXp, 
OH 

Thvniinic  acid  differs  from  the  nucleinic  acids  proper  in  its  ready 
solubility  in  cold  water,  and  in  the  fact  that  it  is  not  precipitated 
from  its  solutions  by  the  mineral  acids.  Like  the  nucleinic  acids,  it 
gives  a  precipitate  with  albumins  or  primary  albumoses  in  acetic 
acid  solution,  but,  in  contradistinction  to  the  nucleinic  acids,  this 
precipitate  is  soluble  in  hydrochloric  acid  and  in  solutions  of  many 
salts. 

The  thyminic  acid  radicle  contains  the  carbohydrate  group  or 
groups  of  the  nucleinic  acid  molecule  ;  the  entire  amount  of  phos- 
phoric acid  ;  a  methyl-uracil  complex  ;  and  in  some  possibly  a  simple 
uracil,  besides  other  bodies  which  are  still  unknown.  The  relation 
of  the  nucleinic  acids  to  the  nucleins  and  its  various  decomp  isition- 
T^roducts  can  accordingly  be  represented  by  the  following  schema  : 

Xiiflein 


Albumin 


Nucleinic  acid 


Xantbin-bases 
and  c^tosin 


Tbvniinic  acid 

Unknown  bodies 


Carbohydrate 
complex 


Phosphoric  acid 


Tbymin, 
viz.,  uracil 

Possibly,  however,  the  decom])Osition  does  not  proceed  in  so 
simple  a  manner  in  all  cases.  Osborne  has  thus  shown  that  on  l)oil- 
ing  tritico-nucleinic  acid  with  dilute  acids  it  loses  about  22  per  cent, 
of  its  phosphorus  as  orthophosohoric  acid,  and  there  remains  a  prod- 
uct which  appears  to  be  analogous  to  thyminic  acid.  It  contains 
one  pentose  group  less  than  the  original  molecule  and  no  longer 
yields  guanin  and  adenin,  as  these  also  have  been  s])lit  off.  Its 
formula,  determined  by  deduction  of  these  radicles  from  the  original 
molecule,  would  l)e  C^^jH^-X.PoO^^^. 

Sehmiedel)erg's  nucleotin-]>hosphoric  acid  represents  the  corre- 
sponding thyminic  acid  of  salmonucleinic  acid. 

The  plasmiuic  acid  which  Kossel  and  Ascoli  obtained  from  yeast 


THE  NUCLEINIC  ACIDS.  99 

nucleinic  acid  apparently  represents  a  product  intermediary  between 
tliyniinic  acid  and  the  original  substance.  Its  formula  is  given  as 
Ci,H2,^N,;P,.0..„.  It  still  contains  nucleinic  bases,  one  or  more  carbo- 
hydrate groups,  and  possibly  ammonia.  According  to  Ascoli,  the 
substance  also  contains  iron  in  masked  form. 

The  phosphorus  of  the  nucleinic  acids  is  possibly  present  in  a  form 
analogous  to  the  polymetaphosphoric  acids ;  Schmiedeberg  suggests 
pyrophosphoric  acid  as  a  possibility. 

According  to  Kossel,  at  least  two  different  carbohydrate  complexes 
may  occur  in  the  nucleinic  acids,  viz.,  1,  a  pentose,  which  has  been 
shown  to  be  /-xylose,  first  demonstrated  in  yeast  nucleinic  acid  and 
later  shown  to  be  a  common  complex  of  the  various  nucleinic  acids  ; 
2,  a  reducing  hexose,  which  on  decomposition  yields  laevulinic  acid 
and  formic  acid  ;  this  was  found  in  the  thymonucleinic  acid,  and 
later  in  that  obtained  from  the  spermatozoa  of  the  sturgeon. 

Whether  or  not  all  nucleinic  acids  contain  a  pentose  grouj)  has 
been  rendered  somewhat  doubtfid  by  Bang's  statement  that  tiiis  is 
absent  in  thymo-nucleinic  acid.  He  adds  that  the  thymus  itself 
contains  considerable  quantities  of  a  pentose.  In  tritico-nucleinic 
acid  Osborne  and  Harris  obtained  evidence  of  three  pentose  groups, 
while  a  hexose  was  absent. 

The  basic  decomposition-])roduets  of  the  nucleinic  acids,  as  has 
been  mentioned,  are  the  common  purin  bases — xanthin,  hypoxanthin, 
guanin,  and  adenin  ;  and  the  pyrimidin  derivatives  cytosin,  uracil, 
and  thymin.     These  will  be  considered  below. 

There  remains  an  unresolved  residue,  of  which  nothing  is  known. 
To  what  extent  this  is  concerned  in  the  production  of  melanins  or 
melanoid  substances,  which  are  constant  dccomposition-])ro(lucts  of 
the  nucleinic  acids  (Schmiedeberg,  Osborne,  and  Harris),  remains  to 
be  seen. 

The  Pyrimidin  Derivatives  of  the  Nucleinic  Acids. 

The  j)yrimidiu  derivatives  of  the  nucleinic  acids  are  cytosin, 
uracil,  and  thymin. 

Cytosin. — Cytosin  is  apparently  a  constant  decomposition-product 
of  all  nucleinic  acids.  It  lias  been  obtained  from  thymo-nucleinic 
acid,  from  the  spermato-nucleinic  acid  of  the  sturgeon  and  herring, 
from  the  nucleinic  acid  of  the  spleen,  the  liver  etc.  Its  formula  is 
C4H5N3O  ;  structurally  it  is  6-amino-2-oxypyrimidin  : 

N==rNH 

I  I 

CO         CH 

I  II 

NH CH 

If  we  compare  with  this  the  structure  of  tlie  purin  bases,  and 
notably  of  uric  acid,  which  is  a  constant  oxidation-product  of  the 
purin  bases  and  is  formed  synthetically  from  pyrimidin  derivatives 


100      THE  XITEOGEXOUS  DERIVATIVES  OF  THE  ALBUMINS. 

(see  Uric  Acid),  the  intimate  relation  which  exists  i)et\veen 
pvrimidin  and  cytosin,  on  the  one  hand,  and  purin  and  the  puriu 
derivatives  on  the  other  is  at  once  apparent.  It  is  quite  likely 
indeed  that  cytosin  represents  the  mother-substance  of  the  pnrin 
group.     These  relations  are  expressed  by  the  following  formulae  : 

(3)X C(6)  N==:C 

ii  II  I  I 

(2)C         C(5)  C        C-N^ 

(1)X=C(4)  N C— N^ 

Pyrimidin  radicle.  Puriu  radicle. 

N=^C.XH2  HX CO 

I         I  I  I 

CO     CH  CO      C^XHx 


CO 


HX CH  HX C-XH" 

Cytosin.  Uric  acid. 


Reactions. — Xitrous  acid  transforms  cystosin  into  uracil,  according 
to  the  equation  : 

C.H^X.O  -  HXO2  =  C.H.X^Oj  +  2X  ^  HjO 

Cytosin.  Uracil. 

On  oxidation  with  barium  permanganate  cytosin  yields  biuret 
and  oxalic  acid  : 

C.H^X^O  —  40  ^  B..p  =  CJH5X3O2  -f  C^Hp, 
Cytosin.  Biuret  Oxalic  acid. 

Like  many  other  pyrimidin  derivatives,  cytosin  on  treating  Avith 
freshly  prepared  chlorine  water  and  the  subsequent  addition  of  a 
trace  of  ammonia  and  the  application  of  heat,  gives  a  red  color 
(murexid  reaction). 

On  treating  a  dilute  solution  of  cytosin  with  a  few  drops  of  a 
concentrated  neutral  solution  of  silver  nitrate,  needles  of  a  double 
salt  crystallize  out  on  standing,  which  are  soluljle  with  difficulty  in 
cold  water  and  resemble  kreatinin  silver  nitrate  in  appearance. 

Uracil. — It  has  been  pointed  out  in  the  foregoing  section  that 
cytosin  yields  uracil  on  treating  with  nitrous  acid.  It  has  therefore 
been  suggested  that  uracil  ])ossibly  does  not  occur  preformed  in  the 
nucleinic  acid  complex,  but  is  merely  a  secondary  product.  Osborne, 
who  first  encountered  uracil  among  the  decomposition-jiroducts  of 
tritico-nucleinic  acid,  expressed  the  opinion,  that  possibly  the  sub- 
stance might  occupy  an  analogous  po.sition  in  the  vegetable  world  as 
thymin  does  in  animal  tissues.  As  a  matter  of  fact  Ascoli  could 
also  demonstrate  uracil  among  the  cleavage-])roducts  of  yeast, 
nucleinic  acid.  More  recently,  however,  Kosscl  and  Stcudel  also 
obtained  the  substance  from  thymo-nucleinic  acid  and  the  spermato- 
nucleinic  acid  derived  from  the  testicles  of  the  herring. 

Structurally  it  is  2.6-dioxypyrimidin  ;  its  relation  to  cytosin  is 
apparent  from  the  formulae  : 


THE  NUCLEI  NIC  ACIDS.  101 

N=C.XH2  H^' CO 

II  I          I 

OC        CH  OC        CH                      QH.y.O^ 

I  II  I            II                                 l-'racii: 
HX CH                    HX CH 

Cytosin.  Uracil. 

Thymin. — Thymin  has  been  obtained  from  thynio-nucleinic  acid, 
sperniato-nucleinic  acid,  the  nucleinic  acids  of  the  liver  and  the 
spleen,  etc.  It  is  generally  regarded  as  a  decomposition-product 
of  the  thymiuic  acid  complex,  but  it  has  not  yet  been  definitely 
established  that  a  thymin  radicle  is  present  in  all  nucleinic  acids. 
It  is  supposedly  absent  in  guanylic  acid.  The  amount  which  can 
be  obtained  is  variable ;  some  nucleinic  acids  apparently  furnish 
more  cytosin,  others  more  thymin. 

Like  uracil,  thymin  is  a  pyrimidin  derivative.  It  is  5  methyl,- 
2.6-dioxypyrimidin  and  thus  isomeric  with  5,-methyl  uracil. 

HX CO  HX CO 

II  II 

OC        CH  OC         C CH3  C5HgX,02 

I            11                                   I  I  Thymin. 

HX CH  HX CH 

Uracil.  Thymin 

(methyl-nracil). 

As  a  pyrimidin  derivative  thymin  is  closely  related  to  the  piirin 
bases  and  uric  acid  (see  the  following  pages).  Isodialuric  acid  f(n' 
example,  which  is  closely  related  to  uracil,  can  be  readily  condensed 
with  urea  to  uric  acid,  as  shown  in  the  equation  : 

C,H,X,0,  ^  CO(XH,),  =  C,H,X,03  -  2H,0 
Dialuric  acid.  trea.  I  rie  acid. 

The  Purin  Derivatives  of  the  Nucleinic  Acids. 

The  common  purin  derivatives  of  the  nucleinic  acids  are  xanthin, 
hypoxanthin  (.sarcin),  guanin,  and  adenin.  Derivatives  of  these  in 
turn  are  heteroxanthin,  paraxanthin,  theophyllin,  theobromin,  caf- 
fein,  epiguanin  (episarcin),  and  carnin.  Of  these,  paraxanthin, 
heteroxanthin,  and  epiguanin  (episarcin)  have  thus  far  only  been 
found  in  the  urine.  Theophyllin,  theobromin,  and  caff'ein  only 
occur  in  the  vegetable  world,  wliile  the  remaining  members  of  the 
group  ai'e  common  con.^tituents  of  both  animal  and  vegetable  cells. 
Collectively  tiiey  are  termed  purin,  xanthin,  or  alloxuric  bases. 
They  are  all  closely  related  to  each  other  and  to  uric  acid,  which,  as 
we  shall  see  later,  is  one  of  the  most  important  end-products  of 
nitrogenous  metabolism  in  the  animal  world.  They  are  all  deriva- 
tives of  E.  Fischer's  hyjiotiietical  purin,  and  in  tliis  manner  closely 
related  to  the  pyrimidin  derivatives  just  considered  in  the  forego- 
ing section.  Adenin,  guanin,  xanthin,  and  hypoxanthin  are  direct 
derivatives  of  the  nucleinic  acids  and  possibly  of  the  cytosin  com- 
plex (see  above). 


102      THE  NITROGENOUS  DERIVATIVES  OF  THE  ALBUMINS. 

Their  structural  relations  to  each  other  and  to  purin  are  shown 
below  : 

(6) 

(1)  y — CH 


I  I  (7) 

(2)HC      (5)C NHx 

II  II 
(3)  N C N 

(4)  (9) 


^CH(8)  P„rin  =  CjH.X,. 


By  substituting  the  group  NH2  for  the  hydrogen  atom  at  6,  adenin 
results,  and  is  hence  spoken  of  as  a  6-amino-purin  : 

N=C.NHj 

I  I 

HC         C NHv 

!!  li  >CH  Adenin  =  C5H5N5. 

Hy])0xanthin,  according  to  this  conception,  would  be  6-oxypnrin, 
xanthin  2,  6-dioxypurin,  and  guauin  2-amino-6-oxypurin  : 

HX CO 

I            I 
CO         C XH. 

I  11  >CH 

N C X^  HX C X^ 

Hypoxanthin  =  C6H4N4O.  Xanthin  =  C5H4N4OJ. 

HX CO 

I             I 
HX=C  C XH. 

I  If  }cu 

HX C X  ^ 

Guanin  =  f'sHsXjO. 

From  these  jjrimary  bodies  the  remaining  ones  then  result  through 
a  substitution  of  methyl  groups.  Heteroxanthin  is  thus  formed 
from  xanthin  by  replacing  the  hydrogen  atom  at  7  with  CH^,  and 
is  therefore  7-methyl-2, 6-dioxypurin.  Paraxanthin  is  accordingly 
l,-7-dimethyl-2, 6-dioxypurin,  and  caffein  1,3, 7-trimcthyl-2, 6-dioxy- 
purin. Theophyllin  and  theobromin  are  isomeric  with  paraxanthin, 
and  structurally  l,3-dimethyl-2, 6-dioxypurin  and  3,7-dimethyl-2,6- 
dioxypurin,  respectively.  Epiguanin  is  similarly  derived  from 
guanin  by  the  replacement  of  the  hydrogen  atom  at  7  with  methyl, 
and  is  hence  7-methyl-guanin. 

HX CO  CH3.X CO 

I  I  II 

CO         C X.CH,v  CO        C X.CH3 

I  I  ^-CH  I  ji  ^  CJl 

HX C X^^^^  HX C X  ^^ 

Heteroxanthin  =  CcHeN402.  Paraxanthin  =  C7H8N4O2. 

CH3.X CO 

'II 

CO        C X.CH3. 

i  I  _JCH 

CH3.X — c — x^===^ 

CaflFein  =  C8H10X4O2. 


THE  NUCLEINIC  ACIDS.  ■  103 

CH3.N CO  HN CO 

CO        C NH\  CO        C N.CH3. 

I  II  >CH  I  II  ^CH 

CH3.N C N    ^  CH3.N C N^===^ 

Theophyllin  =  C7H8N4O2.  Theobromin  =  CjHgNiOa. 

HN CO 

I  I 

HN  =  C         C N.CH3. 

I            il  _^CH 

HN C N.:^=^ 

Epiguanin  =  C6H7N6O. 

Carnin  is  apparently  closely  related  to  hypoxanthin,  as  on  treatment 
with  bromine  it  yields  methyl  bromide,  carbon  dioxide,  and  the  brom- 
hydrate  of  hypoxanthin,  according  to  the  equation  : 

C,H8NA  +  2Br  ^  CjH.N.O.HBr  +  CHjBr  +  CO^. 
Its  structnral  formula  is  thus  possibly  the  following : 

CH3.N CO 

I.  I 

CHOH  C NH  \ 

I                      II  ^CH 

HN— CO  =  CO N  ^:^ 

Episarcin  is  but  little  known.  Its  formula  is  given  as  C^HgNgO, 
but  it  is  possible  that  this  is  not  correct.  The  substance  has  un- 
doubtedly many  properties  in  common  with  epiguanin,  and  future 
researches  indeed  may  possibly  show  that  the  two  are  identical. 

These  various  nucleinic  bases,  which  Gautier  also  designates  as 
the  xanthin  leucomains,  have  the  character  of  feeble  alkaloids,  and 
combine  with  hydrochloric  acid  and  platinum  chloride  to  form 
crystalline  salts,  which  are  dissociated  only  very  slowly  or  not  at  all 
by  water.  When  fused  with  alkalies  they  lose  the  greater  portion 
of  their  nitrogen  in  the  form  of  cyanogen,  and,  as  a  matter  of  fact, 
we  find  that  all  of  them  contain  the  group  HCN.  Adenin,  indeed, 
is  a  polymeric  compound  of  liydrocyanic  acid,  and  xanthin  can  be 
obtained  by  direct  hydration  of  the  same  body  (Gantier).  On  heat- 
ing with  alkalies  or  with  water  the  nucleinic  bases  generally  do  not 
give  rise  to  the  formation  of  lu-ea,  and  they  cannot  hence  be  re- 
garded as  ureids,  although  a  close  rekitionship  exists  between  them. 
By  a  simultaneous  process  of  oxidation  and  liydration  guanin  thus 
gives  rise  to  parabanic  acid,  and  it  is  possible  indeed  to  pass  from 
guanin  to  xanthin  and  hypoxanthin  by  a  series  of  simple  reactions 
without  tlie  intervention  of  urea.  These  changes  are  represented  by 
the  equations  : 

/NH2       CO— NH. 
C^H^N^O  +  30  +  HP  =  C(NH)<  +    |  /CO  +  CO, 

Ciuanin.  ^NH,        CO— NH-^ 

Guanidin.  Parabanic  acid. 

CsHsN.O  +  HNO2  =  C.H.N^,  +  2N  +  H,0. 
Guanin.  Xanthin. 

CH.NA  +  2H  =:.  C^H^Np  +  H,0. 

Xanthin.  Hypoxanthin. 


104      THE  NITROGENOUS  DERIVATIVES  OF  THE  ALBUMINS 

Xanthin,  however,  on  oxidation  and  hydration  yields  urea  and 
alloxan,  Avhieh  latter  is  further  oxidized  to  parabanic  acid  and 
carbon  dioxide,  as  shown  in  the  equations  : 

CO-NH. 

I  \  /NH, 

C,H,N,0,  -1-  20  +  HP  =  CO  >C0  +  C0< 

Xauthin."  I  /  \NH, 

CO— NH^  Urea. 

Alloxan. 

CO— NH. 

I  \  CO— NHv 

CO  >C0  +  0=1  >C0  +  CO, 

I  /  CO— NH/ 

CO— NH^ 

Alloxan.  Parabanic  acid. 

The  close  relationship  which  exists  between  uric  acid  and  the  nucle- 
inic  bases  thus  becomes  apparent,  as  uric  acid  on  oxidation  and 
hydration  gives  rise  to  the  same  products  as  xanthin : 

CsH^N.O.,  +  O  +  H.,0  =  C4H2NA  +  CON^H,. 
Uric  acid.  Alloxan.  Urea. 

This  relationship  is  further  shown  by  decomposing  the  primary 
xanthin  bases  and  uric  acid  with  fuming  hydrochloric  acid  or 
hydriodic  acid  under  high  pressure.  Qualitatively  the  same  prod- 
ucts are  thus  obtained,  viz.,  ammonia,  carbon  dioxide,  glycocoll, 
and  formic  acid,  while  the  quantitative  relations,  of  course,  vary 
with  the  nature  of  the  individual  substance. 

C5H5N5      +  8H2O  =  4NH3  +    CO2  4-  CH.,.NH2  COOH  +  2H.C00H. 
Adenin.  Glycocoll.  Formic  acid. 

C,H,N,0   +  7H2O  =  3NH3  +    CO2  +  CH2.NH2.COOH  +  2H.C00H. 
Hypoxanthin. 

C5H5N-O   +  7H2O  =  4NH3  +  2C0,  +  CH,,.NH2.C00H  +  H.COOH. 
Guanin. 

C,H,N,02  +  6H2O  =  3NH3  +  2C0j  +  CH2.NH2.COOH  +  H.COOH. 
Xanthin. 

C5H,N,03  +  5H2O  =  3NH3  +  3CO2  +  CH.,.NH.,.COOH. 
Uric  acid. 

Accordino-  to  Emil  Fischer,  the  structural  formula  of  uric  acid  can 
be  represented  as  follows  : 

HN  —  CO 

I        II 
CO    C— NH. 

I        I  >C0 

HN— C— NH^ 

Uric  acid. 

and  it  is  thus   seen   that,  like  the   nucleinic  bases,  it  contains  the 
purin  radicle. 

All  the  nucleinic  bases  combine  with  acids  and  alkalies  to  form 
salts,  many  of  which  are  readily  crystallizable.  On  boiling  with 
acetate  of  copper  most  of  them  are  thrown  down  as  in.soluble  com- 
pounds.    From  their  neutral  solutions,  or  in  the  presence  of  a  little 


THE   UREIDS.  105 

ammonia,  they  are  precipitated  by  ammoniacal  silver  nitrate  solu- 
tion. This  precipitate  dissolves  in  nitric  acid  on  the  application  of 
heat,  but  reappears  on  cooling.  Most  of  the  nucleinic  bases,  more- 
over, when  evaporated  to  dryness  in  the  presence  of  nitric  acid, 
leave  a  yellowish  residue,  which  changes  to  orange  and  often  to  a 
temporary  purple  on  the  addition  of  an  alkali.  In  this  respect  also 
these  substances  resemble  uric  acid,  which  gives  a  very  similar  reac- 
tion (see  Murexid  test).  When  exposed  to  the  action  of  putrefactive 
organisms  adenin  is  transformed  into  hypoxanthin,  and  guanin  into 
xanthin,  so  that  these  two  only  are  found  in  decomposed  material. 

For  a  description  of  the  individual  members  of  this  group  which 
are  found  in  the  animal  body,  as  well  for  the  method  of  their  isola- 
tion and  quantitative  estimation,  the  reader  is  referred  to  subsequent 
chapters  (see  especially  pages  259  and  384). 

THE  UREIDS. 

The  ureids  comprise  a  number  of  nitrogenous  crystallizable  bodies 
characterized  by  the  fact  that  on  hydroly tie  decomposition  alone,  or  on 
simultaneous  oxidation,  they  yield  urea.  They  are  hence  derivatives 
of  urea,  and  may  contain  one  or  more  molecules  of  this  substance,  in 
which  one  or  more  hydrogen  atoms  are  replaced  by  radicles  of  mono- 
basic or  polybasic  acids.  On  decomposition  they  yield  either  urea 
and  a  non-nitrogenous  acid  directly,  or  they  give  rise  to  urea  and  a 
less  complex  ureid,  which  is  then  further  decomposed,  as  in  the 
first  instance.  Thev  are  accordingly  divided  into  mono-ureids  and 
di-ureids.  The  former  generally  contain  two  atoms  of  nitrogen  in 
their  molecule,  while  the  latter  possess  four  atoms  of  nitrogen.  All 
these  bodies  are  closely  related  to  each  other  and  to  the  nucleinic 
bases,  from  which  they  are,  in  part  at  least,  derived. 

The  mono-ureids  comprise  alloxan,  alloxanic  acid,  dialuric  acid, 
barbituric  acid  or  malonyl-urea  and  its  derivatives,  all  of  which  can 
be  decomposed  into  urea  and  non-nitrogenous  acids  containing  three 
atoms  of  carbon.  From  these  another  series  of  mono-ureids  is  de- 
rived, which  also  yield  urea  and  non-nitrogenous  acids  ;  but  the 
latter,  in  contradistinction  to  the  first  group,  contain  only  two  atoms 
of  carbon.  These  include  parabanic  acid,  oxaluric  acid,  allanturic 
acid,  and  hydantoin  and  its  derivatives,  viz.,  hydantoic  acid  or  glyco- 
luric  acid,  and  methyl-hydantoin.  The  di-ureids  are  similarly 
divided  into  two  groups,  the  first  of  which  comprises  uric  acid, 
alloxantin,  murexid  or  ammonium  purpurate,  and  hydurilic  acid  ; 
while  the  second  is  represented  by  allantoin,  allantoic  acid,  and 
allituric  acid. 

The  best  known  representative  of  the  ureids  is  uric  acid.  From 
it  all  others  can  be  derived,  and  a  description  of  its  general  proper- 
ties and  reactions  may  serve  as  an  illustration  of  the  entire  class. 

Uric  acid  is  a  crystallizable  dibasic  acid  of  the  formula  C^H^N^Og. 
Structurally  it  may  be  regarded  as  a  purin  derivative,  and  may  be 
represented  by  the  formula  : 


106      THE  NITROGENOUS  DERIVATIVES  OF  THE  ALBUMINS. 

HN-CO 

CO   C— XH\ 

I       II  )C0. 

HN — C— NII^ 

It  was  first  obtained  synthetically  by  treating  a  mixture  of  glycocoll 
and  urea  at  a  temperature  of  230°  C.  until  the  melted  mass  assumed 
a  yellowish  color  and  became  turbid.  The  reaction  which  took  place 
may  be  represented  by  the  equation  : 

CH2.NH2.COOH  +  3CO(NH2)2  =  CjH.NA  +  2H2O  +  3NH3 

Glycocoll.  Urea.  Uric  acid. 

The  same  result  may  be  reached  by  heating  a  mixture  of  urea  and 
trichlorolactic  amide : 

C.,H,Cl.jXO,  +  2CO(NH2).,  =  C5H,N403  +  H^O  +  HCl  +  NH4CI 
Trichlorolactic  amide. 

As  indicated  in  the  previous  section,  moreover,  uric  acid  also  results 
through  the  condensation  of  isodialuric  acid  and  urea,  according  to 
the  equation  : 

HN— CO  HN-CO 

II  II 

OC  C(OH)+  COCNH^)^  =     CO  C— NH. 
I       II  I        II  >C0  +  2H.p 

HN     C(0n)  HN— C— NH^ 

When  treated  in  the  dry  state  uric  acid  is  decomposed  into  hydro- 
cyanic acid,  cyanuric  acid,  ammonium  carbonate,  ammonium  cyanate, 
biuret,  etc.  Fu.sed  with  an  exce.^^s  of  potassium  hydrate,  it  similarly 
yields  ammonia,  potassium  carbonate,  oxalate,  cyanate,  and  cyanide. 

On  reduction  with  nascent  hydrogen,  in  the  presence  of  water  and 
sodium  amalgam  uric  acid  is  first  transformed  into  xanthin,  and  sub- 
sequently into  hypoxanthin.  The  close  relationship  which  exists 
between  the  nucleinic  bases  and  uric  acid  is  thus  further  shown 
(see  page  104). 

Analogous  to  the  synthetic  formation  of  uric  acid  from  urea  and 
glycocoll,  we  find  that  on  decomposition  with  hydriodic  acid  the 
substance  yields  carbon  dioxide,  ammonia,  and  glycocoll,  viz.,  the 
same  products  which  are  obtained  from  the  nucleinic  l)ases. 

From  the  reactions  which  have  thus  tar  been  described  the  nature 
of  uric  acid  as  a  ureid  is  not  apparent.  If  the  hydrolytic  decom- 
position of  the  substance  is  effected  less  energetically,  this  becomes 
manifest  at  once.  On  prolonged  boiling  with  water  it  is  decom- 
posed into  urea  and  dialuric  acid,  which  latter  further  yields  urea 
and  tarfronie  acid.  In  this  manner  the  character  of  uric  acid  as  a 
diureid  of  the  first  order  is  demonstrated.  The  reactions  which 
take  place  are  represented  by  the  equations : 

(1)  CsH.NiOs  +  2H2O  =  C,H,NA  +  C0(NH.,^2 

Uric  acid.  Dialuric  acid.         Urea. 

(2)  C,H,N.A  +  2H,0  =  C3HA       -fCOlNH,), 
Dialuric  acid.  Tartronic  acid.  Urea. 


THE   UREIDS.  107 

On  treating  with  an  oxidizing  agent,  in  the  presence  of  water,  uric 
acid  is  similarly  decomposed  into  urea  and  the  mono-ureid  alloxan, 
which  can  be  further  decomposed  into  urea  and  mesoxalic  acid  : 

(1)  CjH.N.Os  +  H,0  +  O  -  C,H,XA  +  COINH^), 
Uric  acid.  Alloxan. 

(2)  C,H2N.A  +  2H,0         =C3HA      +  CO(NH,), 

Alloxan.  Mesox'alic 

acid. 

Its  relation  to  the  di-ureids  of  the  second  order  is  shown  by  oxidiz- 
ing the  substance  with  peroxide  of  manganese  in  neutral  solution  at 
a  moderate  temperature.  In  this  manner  allantoin  is  formed,  from 
which,  on  further  oxidation,  urea  and  oxalic  acid  result : 

(1)  CsH^N.Oj  +  H.p     +  O  =  C,HeN,03  +  CO, 

Allantoin. 

(2)  C.HeNA  +  2H,0  +  O  =  C^H^O,  +  2C0(NH,), 

Oxalic 
acid. 

Of  special  interest,  further,  is  the  formation  of  mnrexid,  or 
ammonium  purpurate,  which  results  when  uric  acid,  even  in  minimal 
amounts,  is  evaporated  together  with  nitric  acid,  and  the  reddish 
residue  is  brought  in  contact  with  ammonia.  A  beautiful  purplish- 
red  color  then  develops,  which  is  characteristic  of  uric  acid  and  its 
salts  (murexid  test).  The  reactions  which  take  place  may  be  rep- 
resented by  the  equations  : 

(1)  CjH.NA  +2H2O        =C,H,NA  +CO(NH2)2 

Diahiric  acid. 

(2)  C,H,NA  +  NH.OH    =  C,H3(NH,)NA  +  H,0 

Ammonium 
diahirate. 

(3)  2C,H,(NH,)NA  +  0  =  QH,! XHJNA  +  3H.,0 
Ammonium  dialurate.  Murexid. 

The  relation  of  uric  acid  to  the  mono-ureids  of  the  second  order, 
finally,  is  shown  by  treating  one  part  of  uric  acid  with  three  parts 
of  nitric  acid  (50  per  cent,  solution),  and  heating  to  70°  C.  On  sub- 
sequent evaporation  to  a  syrup  and  cooling,  parabanic  acid  crystal- 
lizes out,  and  on  decomposition  yields  urea  and  oxalic  acid  : 

(1)  C.H.NA  +  H,0  +  O  =  C,H,NA  +  CO  (NH,), 

Alloxan. 

(2)  C,H,NA  +  O  =  C,H,N A  +  CO, 

Parabanic 
acid. 

(3)  C3H,,NA  +  2H,0         =C.,HA      +  CO(NH,), 

Oxalic  acid. 

Guanin,  as  has  been  shown,  under  the  same  conditions  yields 
guanidin  and  parabanic  acid,  which  further  illustrates  the  close 
relationship  between  the  two  classes. 

For  a  consideration  of  the  methods  employed  in  the  isolation  of 
uric  acid  and  its  quantitative  estimation,  tlie  reader  is  referred  to  the 
chapter  on  the  Urine. 


108      THE  NITROGEyOrS  DERIVATIVES   OF  THE  ALnVMISS. 

THE   KREATINS. 

The  kreatins,  or  hreatinic  /eucomains,  as  they  are  also  termed  by 
Gautier,  are  basic  substances  which  are  closely  related  to  the 
nuclcinic  bases  and  to  the  ureids,  Avhich  have  just  been  considered. 
They  comprise  kreatin,  kreatinin,  crusokreatinin,  xanthokreatinin, 
amphikreatinin,  and  two  similar  substances  of  doubtful  composition. 
Ktratin,  moreover,  is  related  to  arginin,  and  can  lie  produced  syntheti- 
cally throuo-h  the  union  of  cyanamide  and  methyl-glycocoll,  as  arginin 
results  from  cyanamide  and  ornitliin.  AVhile  arginin,  however,  can 
be  obtained  artificially  from  albuminous  material,  kreatin  has  thus 
far  not  been  isolated  in  this  manner.  Its  formation  from  cyanamide 
and  methyl-glycocoll  is  represented  by  the  equation : 

N  =  C  — NHj  +  NH(CH3)  —  CH^—  COOH  =  NH  :  C< 

^NCCHg)  —  CH2— COOH 
Cyanamide.  Methyl-glycocoll.  Kreatin. 

It  is  thus  a  homologue  of  glycocyamin,  which  results  through  the 
union  of  cyanamide  and  glycocoll : 

/NH.,  /XH.3 

N=C  —  NHj  +  CH,.  =  NH  :  C< 

'    COOH  \XH  — CH.,  — COOH 

Cyanamide.  Glycocoll.  Glueocyamiu. 

On  dehydration  kreatin  loses  one  molecule  of  water  and  is  trans- 
formed into  kreatinin,  which  may  thus  be  regarded  as  the  anhydride 
of  kreatin  : 

/NH  /NH  CO 

NH  :  C<  =  NH  :  c(  -  H^O 

\N(CH3)  —  CH,  —  COOH  \N(CH3)  —  CH^ 

On  oxidation  with  mercuric  oxide  kreatin  gives  rise  to  the  forma- 
tion of  the  oxalate  of  methyl-guanidin  or  methyl-uramin,  which  in 
turn  results  from  guanidin,  and  this  from  guanin. 

2C,HgXA  +  50  =  (C2H,N3)2.C.,H.,04  +  2C0,  +  H,0 
Kreatin.  Methyi-guanidin 

oxalate. 

On  decomposition  with  baryta-water  kreatin  yields  urea,  methyl- 
glycocoll,  and  a  small  auKjunt  of  hydantoic  acid.  Kreatinin  is 
similarly  transformed  into  methyl-hydantoin,  which  is  then  likewise 
decomposed  into  urea  and  methyl-glycocoll,  thus  demonstrating 
the  close  relationship  which  exists  between  the  kreatins  and  the 
ureids. 

.NH^  CH,  —  XH.CH3 

NH  :  C^  +  H,0  =    I  -  COfXH^), 

^NfCH.,)  —  CH2  —  COOH  COOH  Urea. 

Kreatin.  Methyl-glycocoll. 

/NH CO  /NH CO 

NH  :  C<  I      +  HjO  =  NH3  +  CO.  I 

\N(CH3)  — CHj  \N(CH3)  — CH, 

Kreatinin.  Methyl-hydantoin. 


THE  PTOMAINS.  109 

Kreatin  and  kreatinin  are  homologous  with  lysatin  and  lysatinin, 
which,  as  has  been  seen,  resnlt  from  albuminous  material  when  this 
is  decomposed  with  boiling  mineral  acids.  While  kreatin,  however, 
contains  but  four  atoms  of  carbon,  lysatin  contains  six,  and  is 
represented  by  the  formula  C^Hj^NgO^.  Like  kreatin  and  kreatinin, 
lysatin  and  lysatinin  yield  urea  on  hydrolytic  decomjio.sition. 

The  individual  representatives  of  the  group  will  be  considered 
later. 

THE  PTOMAINS. 

Tiie  term  ptomain  was  originally  applied  by  Selmi  to  certain 
alkaloidal  bodies  which  are  formed  dnring  the  process  of  albu- 
minous putrefaction.  Gautier  then  extended  its  use  to  include  all 
those  alkaloidal  substances  which  resnlt  from  anaerobic  fermenta- 
tion, as  also  those  which  are  formed  in  the  tissues  of  the  higher 
animals  in  the  absence  of  air,  or  in  the  presence  at  least  of  an  insuf- 
ficient supply  of  oxygen.  In  contradistinction  to  these  substances, 
Gautier  terms  those  alkaloidal  bodies  which  are  formed  during  the 
normal  and  aerobic  life  of  the  tissues  leucomains.  Under  this 
latter  heading,  as  has  been  seen,  he  comprises  the  nucleinic  bases 
and  the  kreatins.  Both  classes  of  substances  are  of  sjiecial  interest 
to  the  physician,  as  their  formation  or  undue  accumulation  in  the 
body  may  give  rise  to  serious  disturbances.  This  is  true  more 
particularly  of  the  ptomains,  some  of  which  are  extremely  toxic. 

Gautier  divides  the  ptomains  into  acyclic  and  cyclic  ptomains, 
some  of  which  are  free  from  oxygen,  while  in  others  this  is  present. 
They  are  all  derived  from  ammonia  by  the  substitution  of  various 
radicles  for  one  or  all  of  the  hydrogen  atoms  of  ammonia,  and  are 
hence  analogous  to  the  amins. 

The  acyclic  ptomains  which  are  free  from  oxygen  comprise  the 
following  substances  : 

Methylamin NH^-CH,     =  CH5N 

Dimethvlamin NHiCHa)^    =  C2H7N 

Trimethylamin ^(CHa),,        =  C3H9N 

Butylamin NH2(C;H9)  =  C^H.iN 

Amylaniin NH2(C5H,i)  =  CgHigN 

Hexylamin NH,(CbH,3)  =  C.H.^N 

Neundin CsHhN^ 

*^aprin      ...._.._ C'sHuN., 

Pentamethvlene-diamin  or  cadaverin NH.,(CH.,)5.NH2 

Etliylene-diamin NH'lG.Hj^NHa 

Tetramethylene-dianiin  or  putrescin      ]SIH2(CH2)4.NH2 

Diniethylene-imide  or  spermin (CH.jIjNH 

Mvdalein '- 

Methyl-guanidin CH4(CH3)N3 


110      THE  SITEOGEyOUS  DERIVATIVES   OF  THE  ALBUMISS. 

The  oxygen-containing  acyclic  ptomains  are  the  following  : 

Cholin   or  trimethyl-oxyethylene-am- 1         ^-(CH3)3.(C,H,.OH).OH  =  C,H„NO, 
monium  hvdrate I  \       i/s  \    i    *         i  o     lo       2 

^ rvdrate  "■^"°^^^'>>"^-^^°-^^-^'"°'^°^"'"  }  N(CH3)3.(C,H3).OH  =  QH^jNO 

Muscarin      .    .    .    ......    .    .    .'  .  X(CH3)3.(CH2 COHi.OH  =  C5H„N03 

Betain  or  oxycholin       ^'(CH3)3.(CH2.COOH).OH  =  Q.^,^^0., 

Mvdatoxin CgHuXOj 

MVdin       C„H,,NO 

Gadinin C;H,gN02 

Metliyl-gadinin       C,,HigN02 

Mytil'otoxin CgHisXO, 

Propyl-glucocyamin C6H13N2O3 

The  remaining  ptomain.s  are  partly  cyclic  and  in  part  not  classified  : 

Collidin  fiso-phenyl-ethyiamin)  ....  C6H5.CH(CH3).NH2  =  C,H„N 

Hydrocollidin C\H,3N 

Parvolin C;,H,3N 

Corindin CioH,5N 

Hydrolutidin       C\H,]N 

Hydrocornidin C,nH,;N 

Scombrin      C,;H3J^N^ 

Morrlniin C]t,H2-N3 

Asellin      C^H^'X, 

Morrhuic  acid f5H3(OH)(C3H6.COOH).NH  =:  C,H,;X03 

Typhotoxin C^Hi^NOj 

Tetanin Ci3H3(jN2*-^* 

Tetanotoxin CjHuN 

Spasmotoxin 

Tyrotoxin 

Pyocyanin 

Pyoxanthin 

Some  of  these  snb.stances  and  their  origin  from  the  albumins  have 
already  been  considered,  and  we  shall  have  further  occasion  to  study 
them  in  greater  detail.  Others  are  scarcely  known,  and  require  no 
further  description  at  this  place.  They  are  all  intimately  related  to 
the  vegetable  alkaloids,  with  which  they  have  many  reactions  in 
common.  They  have  strongly  basic  properties,  and  are  capable  of 
combining  with  acids  to  form  .salts.  Like  the  albumins  fr(jm  which 
they  are  derived,  they  are  precipitated  with  the  chlorides  of  platinum, 
mercur}',  and  gold,  as  also  with  tannic  acid,  picric  acid,  phospho- 
raolybdic  acid,  phosphotung.stic  acid,  etc.  AVith  these  they  form 
well-defined  crystalline  .salts,  which  .serve  for  their  diiferentiation 
from  each  other  and  as  a  basis  for  the  determination  of  their  ele- 
mentary composition.  The  methods  which  are  employed  for  the 
separation  of  ptomains  will  be  considered  in  a  subsequent  chapter. 


CHAPTER    YL 

THE  FERMENTS. 

Ix  the  foregoing  chapters  we  have  considered  in  a  general  way 
the  more  important  characteristics  of  the  three  great  chisses  of 
food-stuffs,  and  have  studied  in  some  detail  also  the  various  decom- 
j)osition-products  to  which  they  give  rise  in  their  passage  through 
the  animal  body.  We  have  also  pointed  out  that  with  few  excep- 
tions the  food-stuffs,  which  the  animal  derives  either  directly  or 
indirectly  from  the  plant,  cannot  be  ntilized  by  the  animal  directly, 
but  that  they  must  previously  undergo  certain  changes,  which  vary 
with  the  character  of  the  individual  sul)stances.  The  native  albu- 
mins must  Hrst  be  transformed  into  albumoses  and  simpler  products; 
the  disaccharides  and  polysaccharides  must  be  inverted  lo  mono- 
saccharides, and  the  fats  must  first  be  decomposed  into  glycerin 
and  fatty  acids.  We  have  seen  also  that  in  the  chemical  laboratory 
these  changes  can,  for  the  most  part,  be  brought  about  through  the 
action  of  superheated  steam,  by  boiling  with  acids  and  alkalies,  etc. 
— that  is,  through  agencies  wliich  manifestly  are  not  at  work  in  the 
living  world.  The  question  therefore  suggests  itself:  Whpt  are  the 
means  at  the  disposal  of  living  bodies  to  bring  about  these  changes? 
This  question  has,  in  a  measure,  been  answered  in  the  introductory 
remarks,  where  it  was  pointed  out  that  the  animal  is  capable  of 
brino;ino-  about  a  laro;e  number  of  analytical  chang-es  bv  means  of 
certain  ferments,  or  enzymes,  which  are  furnished  by  the  animal 
cells  themselves.  At  the  present  time  there  is  a  tendency  indeed 
to  assume  that  most  of  the  vital  phenomena  are  referable  to  the 
action  of  ferments,  and  we  know  as  a  matter  of  fact  that  ferment 
action  is  not  necessarily  only  destructive,  but  may  also  be  construc- 
tive. The  discovery  of  oxidation  ferments  has  thrown  further  light 
on  many  processes  which  were  formerly  referred  to  "  vital "  forces 
inherent  in  the  living  protoplasm.  The  existence  of  such  "  vital " 
forces  has  become  the  more  doubtful  the  more  closely  vital  phenomena 
are  studied.  Only  a  few  years  ago  the  ferments  furnished  by  the 
digestive  glands  were  the  only  known  ferments  in  the  animal  body, 
and  our  knowledge  of  the  mechanism  of  the  various  metal^olic 
processes  was  practically  nil.  To-day  we  know  that  ferments  are 
present  in  probably  every  cell  and  are  intimately  concerned  in  all 
its  manifestations  of  life.  In  the  liver-cell,  for  example,  not  less 
than  a  dozen  different  ferments  have  been  demonstrated.  Still  we 
are  only  on  the  threshold  of  our  knowledge  of  intracellular  chomi- 
cal    processes,  and   there  are   still    many   obscure   problems   which 

111 


112  THE  FERMENTS. 

await  tlioir  final  solution.  That  the  life  of  the  cell  is  not  insepar- 
ably connected  with  the  activity  of  its  ferments  has  been  W(;ll 
shown  by  Biichner :  If  common  yeast  is  placed  in  a  solution  of 
cane-sugar,  this  is  inverted  to  glucose  and  laevulose  by  the  contained 
invertin  ;  at  the  same  time  the  cell  causes  the  further  destruction 
of  these  sugars  witli  the  formation  of  alcohol  and  carbon  dioxide. 
The  fact  that  this  latter  ]>rocess  can  be  prevented  by  means  of 
chloroform,  which  is  a  notable  protoj)lasmic  poison,  while  the  inver- 
sion ])roceeds  as  before,  Avas  formerly  interpreted  as  evidence  of  a 
special  vital  activity  on  the  part  of  the  cell.  Buchner,  however, 
has  demonstrated  conclusively  that  there  is  no  true  cause  for  this 
assumption.  For  on  crushing  the  yeast-cells  completely  he  succeeded 
in  obtaining:  a  fluid  which  could  brine;  about  the  further  destruction 
of  glucose  and  hevulose  exactly  as  in  the  case  of  the  living  cell. 
He  could  show  that  this  action  is  referable  to  an  intracellular  fer- 
ment which  is  termed  zymase.  By  similar  methods  a  large  number 
of  intracellular  ferments  have  been  discovered  in  the  animal  body 
(see  below). 

The  ferments  projier  must  be  sharply  distinguished  from  the  so- 
called  ferment-organisms,  or  organized  ferments,  wdiich  occur  widely 
distributed  in  nature  and  comprise  the  important  groups. of  bacteria, 
blastomvcetes,  and  certain  moulds.  These  are  living  beins-s  them- 
selves,  and  not,  as  the  ferments  proper,  products  of  life.  They 
contain  ferments,  and  manifest  their  special  activity,  in  a  great 
measure,  through  their  ferments  ;  but  they  are  not  ferments  them- 
selves, although  they  are  often  so  called.  In  contradistinction  to 
these  organized  ferments,  the  ferments  proper  are  termed  non- 
organized ferments,  or  enzymes.  They  are  specific  products  of  the 
activity  of  cells,  and  occur  widely  distributed  in  the  animal  and 
vegetable  world. 

For  the  maintenance  of  life,  in  the  case  of  the  higher  plants  at 
least,  the  organized  ferments  are  of  prime  importance ;  for,  as  has 
been  seen,  it  is  through  these  low  forms  of  life  that  the  higher  plants 
are  furnished  their  nitrogen  in  a  form  which  they  can  subsequently 
utilize.  In  their  absence  from  the  soil,  vegetable  life,  such  as  we  see 
it,  could  probably  not  exist.  In  the  gastro-intestinal  tract  of  all 
animals  which  have  been  examined  in  this  direction  innumerable 
bacteria  are  also  found,  and  it  was  long  thought  that  their  presence 
here  served  a  very  definite  purpose,  and  that  animal  life  could  not 
go  on  in  their  absence.  This  view,  however,  has  proved  erroneous, 
as  Nuttall  and  Thierfclder  conclusively  demonstrated.  They  showed 
that  Avhen  a  young  guinea-pig,  for  example,  is  removed  from  the 
mother  animal  by  C^esarean  section  under  strict  aseptic  precautions, 
and  is  sul)sequently  fed  with  sterile  food  ;ind  is  furnished  with  sterile 
air,  it  will  grow  as  well  as  a  control-animal.  AMiile  the  presence  of 
bacteria  in  the  animal  body  is  therefore  not  essential  for  the  main- 
tenance of  life,  and  while  it  is  very  questionable,  indeed,  whether 
their  presence  in  the  alimentary  canal  serves  any  useful  purpose  at 


GENERAL  PROPERTIES  OF  THE  FERMENTS.  113 

all,  we  know,  on  the  contrary,  that  the  introduction  of  certain  forms 
is  directly  harmful,  and  that  some  of  the  normal  inhabitants  of  the 
intestinal  canal  may  under  certain  conditions  develop  distinct  patho- 
genic properties. 

General  Properties  of  the  Ferments. — From  what  has  been 
said,  it  is  clear  that  the  ferments  are  capable  of  manifesting  their 
special  activity  even  after  the  death  of  their  mother-cells,  and  it  is 
noteworthy  that  a  great  many  substances  which  are  distinct  proto- 
plasmic poisons  do  not  interfere  with  the  ferments  themselves.  Such 
substances  are  chloroform,  ether,  thymol,  toluol,  salicylic  acid,  arseni- 
ous  acid,  sodium  fluoride,  boric  acid,  hydroxy lamin,  glycerin,  etc. 
In  the  study  of  the  ferments  these  bodies  are  of  great  importance,  as 
we  are  thus  enabled  to  exclude  the  protoplasmic  activity  of  living 
cells,  and  to  determine  whether  certain  chemical  phenomena  which 
we  observe  in  the  tissues  of  the  body  are  referable  to  the  action  of 
ferments  or  not. 

Other  chemicals  not  only  cause  the  death  of  the  cell,  but  also 
arrest  or  annihilate  the  action  of  the  ferments.  Such  substances 
are  the  bichloride  of  mercury,  carbolic  acid,  and  to  a  less  marked 
degree  other  metallic  salts,  as  also  picric  acid,  tannic  acid,  etc.  The 
mineral  acids  are  variable  in  their  action.  Some  ferments  are 
dependent  upon  their  presence,  others  behave  indifPerently,  while 
still  others  are  destroyed.  Some  ferments  are  capable  of  destroying 
others.  The  activity  of  the  ferments  is  further  decreased  with  an 
increase  of  their  specific  ])roducts  beyond  a  certain  degree.  Absence 
of  water  likewise  inhibits  the  action  of  the  ferments,  but  this  is  at 
once  re-established  when  the  necessary  degree  of  moisture  is  supplied, 
and  it  is  possible  therefore  to  preserve  the  ferments  in  the  dry  state. 
During  the  process  of  drying,  however,  care  must  be  had  that  the 
temperature  does  not  exceed  a  certain  limit.  This  varies  with  the 
different  ferments,  but  it  may  be  stated  as  a  general  rule  that  all 
animal  ferments  are  killed  by  a  temperature  of  75°  C,  while  the 
vegetable  ferments  cannot  survive  a  temperature  of  80°  C.  In  the 
absence  of  moisture,  however,  they  can  apj)arently  withstand  much 
greater  heat,  and  it  is  said  that  dry  trypsin,  pepsin,  and  diastase 
may  be  heated  to  a  temperature  of  from  150°  to  160°  C.  without 
losing  their  activity.  Strong  alcohol  destroys  the  action  of  certain 
ferments,  such  as  pepsin  and  diastase,  while  others,  like  the  fibrin- 
ferment,  are  not  affected  unless  the  contact  is  prolonged. 

The  most  peculiar  pr()])erty  of  the  ferments,  and  the  one  which  is 
characteristic  of  all,  is  the  power  to  bring  about  an  amount  of  chemical 
change  which  is  apparently  out  of  all  proportion  to  the  quantity  of 
the  ferment  present,  while  the  ferment  itself  undergoes  no  apparent 
change.  The  common  pepsin  prej^arations  of  the  market  are  of 
a  strength  that  1  part  by  weight  of  the  pepsin  will  digest  6000 
parts  by  weight  of  coagulated  exg-albumin,  and  Petit  claims  that 
a  preparation  from  his  lal)()rat()ry  was  capable  of  dissolving  even 
500,000  times  its  weight  of  fibrin  in  seven  hours. 


114  THE  FER.VEXTS. 

Tliat  tlic  ferments  themselves  undergo  no  change  while  exerting 
tlieir  specific  action  can  be  readily  shown,  as  it  is  possible  to  re- 
obtain  them  from  the  various  digestive  mixtures  and  to  test  their 
efficacy  as  before. 

The  rapidity  with  -which  the  action  of  ferments  takes  ])lace  is 
often  most  remarkable,  and  is  es]X'cially  well  shown  during  the 
coagulation  of  milk   under  the  influence  of  chvmosin. 

In  order  that  the  ferments  may  exhibit  their  activity  to  best 
advantage  a  definite  temperature  is  necessar^%  which  varies  some- 
what with  the  different  ferments,  but  is  generally  about  that  of  the 
body.  Higher  as  well  as  lower  temperatures  gradually  inhibit  their 
action,  and,  as  has  been  seen,  destroy  it  entirely  when  75°-80°  C.  is 
reached.     Very  low  temperature  has  the  same  effect. 

Oxygen  has  no  effect  upon  the  action  of  most  ferments,  and  they 
thus  show^  a  distinct  difference  from  the  organized  ferments,  which 
are  more  or  less  dependent  upon  its  presence  or  its  absence. 

The  reaction  of  the  medium  in  which  the  ferments  are  to  display 
their  activity  is  very  important,  and  varies  with  the  different  fer- 
ments. Some  of  these,  such  as  pepsin,  can  act  to  advantage  only 
in  an  acid  medium  ;  while  others,  such  as  ptyalin,  require  an  alka- 
line reaction  ;  and  still  others  can  act  in  acid,  alkaline,  and  neutral 
media,  but  exhibit  certain  preferences. 

Generally  speaking,  the  mode  of  action  of  the  ferments  is  specific 
— /.  e.,  certain  ferments  will  only  act  upon  certain  definite  substances. 
Fat-sjilitting  ferments  will  only  act  upon  fats,  diastases  only  upon 
carbohydrates,  proteolytic  ferments  only  upon  albumins.  This 
specificity  is  in  some  instances  very  pronounced  and  is  noticeable 
even  in  individual  meml)ers  of  a  common  group.  The  autolvtic 
ferments,  for  example,  will  not  cause  the  cleavage  of  every  albumin; 
generally  speaking  tlntse  albumins  which  are  foreign  to  the  cell 
wiiich  has  furnished  the  particular  ferment  are  attacked  with  greater 
difficulty  by  the  autolvtic  ferments  of  the  cell.  The  autolvtic 
proteolytic  ferment  of  the  liver  is  thus  incapable  of  hydrolyzing 
the  albumins  of  lung-tissue.  For  the  lytic  action  of  ferments  of 
one  organ  upon  material  which  has  originated  in  another  organ 
Jacoby  has  suggested  the  term  lietcroly^is  in  contradistinction  to 
aidolys'iH. 

It  has  long  been  known  that  the  hydrolytic  decomposition  effected 
by  ferments  is  never  carried  to  an  end,  and  it  is  usually  stated  that 
this  is  owing  to  the  fact  tliat  a  gradual  increase  in  the  production  of 
decomposition-products  inhibits  the  action  of  the  ferments  in  ques- 
tion. In  the  light  of  more  recent  investigations,  however,  this 
explanation  is  not  satisfactory.  It  has  been  demonstrated  that 
maltase,  when  added  to  a  solution  of  maltose,  will  cause  inversion 
of  the  latter  to  glucose.  An  end-reaction  is  then  not  obtained  ;  l)ut 
if  this  solution  is  now  added  to  a  solution  of  glucose  in  turn,  at  a 
time  when  further  inversion  does  not  occur,  it  will  be  noted  tliat  a 
retransformation  of  glucose  to  maltose  takes  place,  which,  however. 


CHEMICAL   COMPOSITION  AND   GENERAL  REACTIONS.    115 

is  likewise  not  complete.  It  thus  appears  that  the  ferment  is  not 
only  caj)uble  of  causing-  the  hydrolytic  deconij^osition,  but  also  the 
synthesis  of  maltose;  but  that  in  so  doing  its  action  ceases  as  soon 
as  a  certain  equilibrium  of  reaction  has  been  established.  This 
reversible  action  on  the  part  of  ferments  is,  of  course,  of  the  greatest 
interest  to  the  physiological  chemist,  in  showing  that  the  complex 
syntheses  which  take  place  both  in  plant  and  in  animal  life  mav,  to 
a  certain  extent  at  least,  be  referable  to  such  action,  and  to  forces 
which  are  })robably  at  work  in  the  non-organized  world. 

Kastle  and  Loevenhart  have  demonstrated  the  reversible  action 
of  lipase  in  the  case  of  ethyl  butyrate. 

Frankland-Armstrong  has  shown  under  E.  Fischer  that  lactase 
also  is  capable  of  synthetic  action  with  a  concentrated  solution  of 
galactose  and  glucose. 

In  the  case  of  the  proteolytic  ferments  similar  conditions  probably 
exist.  It  has  been  shown  that  pepsin,  trypsin,  and  papayotin  when 
added  to  concentrated  solutions  of  albumoses,  will  cause  a  precipita- 
tion of  so-called  plastein,  and  Sawjalow  has  announced  that  he 
succeeded  in  coagulating  such  plastei'ns  by  boiling  in  the  presence 
of  acetic  acid,  which  would  establish  their  true  albuminous  nature. 

Abelous  and  Ril)aut  have  further  shown  that  an  extract  of  renal 
tissue  will  bring  about  the  formation  of  hi])puric  acid  from  benzoyl 
alcohol  and  glycocoll.  By  means  of  yeast  maltase  Emmerling  could 
bring  about  the  formation  of  amygdalin  from  glucose  and  amygdalic 
acid  nitril  glucoside. 

Emulsin  also  has  been  shown  capable  of  synthetic  action,  etc. 

Eurtiier  research  will  show  whether  a  reversible  action  is  common 
to  all  ferments. 

Chemical  Composition  and  General  Reactions. — Of  the  chem- 
ical composition  of  the  ferments  but  little  is  known  that  is  definite. 
This  is  owing  to  the  fact  that  isolation  of  any  one  of  the  ferments  in 
a  chemically  pure  form  has  thus  far  not  been  accomplished.  They 
apparently  contain  nitrogen,  and  are  usually  regarded  as  albumi- 
nous substances ;  but  it  is  still  a  matter  of  doubt  whether  this  is 
actually  the  case,  and  it  is  possible  that  the  supposition  of  their 
albuminous  nature  is  owing  to  their  being  contaminated  with 
albumins. 

Like  the  albumins,  they  are  as  a  class  non-diffusible.  They  are 
soluble  in  water,  and  can  be  precipitated  from  their  aqueous  solu- 
tions by  salting  with  ammonium  sulphate  or  by  the  addition  of 
strong  alcohol. 

When  kept  under  alcohol  for  any  length  of  time  some  of  the  fer- 
ments, such  as  pepsin  and  diastase,  are  rendered  inactive  and  are 
apparently  coagulated,  while  the  activity  of  others,  such  as  the 
fibrin  ferment,  remains  unaffected. 

Characteristic  general  reactions,  which  are  common  to  all  fer- 
ments, are  unknown.  Formerly  it  was  supposed  that  they  all  pos- 
sessed the  power  of  decomposing  hydrogen  peroxide,  but  it  appears 


116  THE  FERMENTS. 

that  this  property  does  uot  belong  to  tlie  ferments  proper,  but  to 
adherent  particles  of  protoplasm.  As  a  matter  of  fact,  it  is  possible 
in  a  number  of  ferments  to  destroy  this  power  of  decomposing 
hydrogen  peroxide  without  influencing  their  specific  activity  in  the 
least.  If  pancreatic  juice  is  thus  heated  to  a  temperature  of  60°  C, 
and  then  allowed  to  cool  to  40°  C,  it  will  be  observed  that  the  fluid 
is  still  capable  of  digesting  albumins  and  of  inverting  starch,  while 
it  has  lost  the  power  of  decomposing  hydrogen  peroxide  entirely. 
Similar  results  may  be  obtained  on  heating  the  dry  ferments  to  a 
somewhat  liigher  temperature,  by  treating  with  alcohol,  or  by  satu- 
rating their  solutions  with  neutral  salts. 

Mode  of  Action. — The  decompositions  which  the  ferments  are 
capable  of  eifecting  in  suitable  media  are  essentially  of  a  hydrolytic 
character.  This  can  be  readily  shown  by  comparing  the  decom- 
position-products to  which  the  ferments  give  rise  with  the  original 
substances,  when  it  will  be  found  that,  practically  without  exception, 
the  former  contain  more  water.  Xasse,  moreover,  could  demon- 
strate a  distinct  increase  in  the  electrical  conductivity  of  watery 
solutions  of  starch,  for  example,  when  these  were  treated  with  dias- 
tase, showing  that  dissociated  molecules  of  water  must  have  been 
present.  As  to  the  manner,  however,  in  which  these  hydrolytic 
phenomena  are  brought  about  we  are  very  much  in  the  dark.  On 
the  one  hand,  we  may  suppose  that  the  molecular  oscillations  Avhich 
take  place  in  the  molecules  of  the  ferments  are  of  such  a  nature 
as  to  bring  about  an  increase  in  the  molecular  oscillations  of  the 
substances  upon  which  the  ferments  exert  their  specific  activity,  and 
that  in  consequence  of  this  increase  in  the  oscillations  the  labile 
equilibrium  of  the  large  albuminous  or  polysaccharine  molecules  is 
disturbed,  whicli  in  turn  would  lead  to  new  combinations  of  atoms 
to  form  molecules  that  are  more  stable,  and  the  oscillations  of  which 
would  be  more  nearly  like  those  of  the  ferments.  According  to 
this  theory,  then,  the  action  of  the  ferments  would  be  what  has  been 
termed  a  katalytic  action,  and  analogous  to  the  katalytic  action  of 
various  metals,  such  as  platinum,  gold,  silver,  etc.,  whicli  in  fine 
suspension  behave  in  very  much  the  same  manner  as  the  ferments 
(see  page  20).  On  the  other  hand,  we  may  suppose  that  the  action 
of  the  ferments  is  an  action  by  contact,  such  that  one  molecule  of 
the  ferment  causes  hydrolytic  decomposition  of  one  molecule  of  an 
albuminous  substance,  for  examj)le,  and  that  this  decomposition  in 
turn  causes  decomposition  of  the  adjacent  albuminous  molecules,  and 
so  on.  The  action  of  certain  ferments,  such  as  the  fibrin  ferment, 
and  that  which  causes  the  coagulation  of  milk,  might  very  well 
be  explained  upon  such  a  basis.  For  here  we  see  a  rapidity  of 
action  wdiich  scarcely  admits  of  any  other  explanation  ;  and  direct 
contact  of  the  ferments  with  all  parts  of  the  surrounding  material, 
moreover,  is  excluded,  as  each  ferment-molecule  must  of  necessity 
be  at  once  surrounded  by  a  layer  of  the  coagulated  albumin. 

Classification. — It  has  been  pointed  out  that  there  are  no  general 


CLASSIFICATION.  117 

reactions  which  are  characteristic  of  all  ferments.  The  ferments 
can  be  separated  into  groups,  however,  which  are  fairly  well  char- 
acterized by  their  specific  activity  and  the  decomposition-products 
to  which  they  give  rise.  They  are  accordingly  divided  into  the 
following  classes  : 

1.  The  Proteolytic  Ferments  (Proteases). — These  comprise  the  com- 
mon digestive  ferments  of  the  stomach  and  pancreas,  viz.,  pepsin 
and  trypsin ;  the  autolytic  ferments  which  are  responsible  for  the 
aseptic  autodigestion  of  the  various  organs  after  death,  and  analo- 
gous ferments  which  are  widely  distributed  in  the  vegetable  world. 
They  all  digest  the  various  albumins  with  the  formation  of  albu- 
moses  and  those  end-products  of  hydrolysis  which  are  collectively 
spoken  of  as  peptones.  While  their  activity  is  generally  speaking 
specific  (see  preceding  page),  it  is  noteworthy  that  some  proteolytic 
ferments  will  also  hydrolyze  the  nucleinic  acids  (Araki) ;  trypsin  is 
a  notable  exception  to  this  latter  rule. 

Closely  related  to  the  proteolytic  ferments  is  the  erepsm  which  O. 
Cohnheim  demonstratcnl  in  the  intestinal  mucosa  ;  its  specific  activity 
is  directed  to  the  hydrolysis  of  albumoses,  while  albumins  are  not 
affected. 

2.  The  Amylolytic  Ferments  (Amylases,  Diastases). — These  include 
the  ptyalin  of  the  saliva,  the  diastatic  ferment  of  the  pancreatic 
juice,  the  so-called  vegetable  diastase  and  related  ferments,  which 
may  be  obtained  from  bacteria.  Some  of  these  only  render  starch 
soluble,  while  others  also  cause  its  hydrolysis  to  monosaccharides. 
To  this  class  also  belongs  a  diastatic  ferment  of  the  liver,  which 
causes  the  formation  of  glucose  from  glycogen. 

3.  The  Inverting  Ferments  (Invertases). — These  are  ajiparently 
related  to  the  amylolytic  ferments,  and  are  to  a  certain  extent  iden- 
tical with  them.  They  invert  the  disaccharides  to  monosaccharides, 
and  according  to  their  specific  effect  upon  cane-sugar,  maltose,  and 
lactose,  they  are  termed  invertins  (invertases),  maltases,  and  lac- 
tases, respectively.  Such  ferments  are  found  in  the  saliva,  the  pan- 
creatic juice,  the  enteric  juice,  in  many  of  the  higher  plants,  and  also 
in  numerous  organized  ferments. 

4.  The  Lipolytic  Ferments  (Lipases). — Lipases  are  extensively  dis- 
tributed in  the  animal  body.  They  occur  in  the  gastric  and  the 
pancreatic  juice,  and  are  possibly  represented  in  all  tissues.  In  the 
vegetable  world  also  thev  are  common  ;  lipases  have  been  found  in 
the  seeds  of  ricinus,  of  the  poppy,  of  cannabis  sativa,  in  linseed, 
corn,  etc.  They  cause  the  hydrolysis  of  fats  to  fatty  acids  and 
glycerin, 

5.  The  urases,  viz,,  ferments  which  decom])ose  urea  with  the  for- 
mation of  ammonia.  Such  ferments  are  present  in  many  bacteria, 
such  as  Micrococcus  urere.  Bacterium  ureae.  Bacillus  fluorescens,  etc. 
A  ferment  of  this  order  has  also  been  demonstrated  in  the  liver. 

6.  Ferments  which  transform  amido-acids  into  amides  occur  in  the 
liver  and  the  kidneys,  and  have  also  been  demonstrated  in  plants. 


118  THE  FERMENTS. 

7.  The  Histozyme  of  Schmiedeberg. — This  is  found  in  the  kidneys 
and  is  eliaracterized  l)v  its  al)ility  to  decom])ose  hippuric  acid  Mitli 
the  formation  of  benzoic  acid  and  glycocoll. 

8.  Ferments  which  cause  the  Cleavage  of  Glucosides. — Such  fer- 
ments are  quite  common  among  invertebrates,  but  are  especially 
abundant  in  the  higher  plants.  Examples  are  theemulsin  or  synap- 
tase  of  iiitter  almonds,  the  my  rosin  of  mustard  seeds,  and  other 
Cruciferce,  etc. 

9.  The  nucleases,  viz.,  ferments  which  are  capaljle  of  cau.-ing  the 
cleavage  of  the  nucleinic  acids. 

All  these  ferments  are  characterized  by  their  hydrolytic  action 
and  must  be  diflPerentiated  from  the  oxidation  ferments  (see  below). 
Occupying  an  intermediate  position  between  the  two  groups  are  : 

10.  Ferments  which  are  capable  of  splitting  off  carbon  dioxide 
from  certain  bodies. — Ferments  of  this  order  are  but  little  known, 
but  their  presence  is  indicated  in  various  ways.  Emerson  has 
shown  that  a  ferment  belonging  to  this  class  occurs  in  the  pancreas, 
and  is  characterized  by  its  ability  to  form  oxyphenyl-ethylamin  from 
tyrosin  by  splitting  off  COj.  A  similar  ferment  apparently  occurs 
in  the  liver  and  may  be  responsible  for  the  transformation  of 
cystin  acid  to  taurin. 

11.  The  Oxidation  Ferments. — These  are  ferments  which  are  inti- 
mately concerned  in  the  endocellular  oxidations.  They  can  be 
divided  into  three  groups. 

(a)  The  Oxygenases. — These  are  supposedly  of  albuminous 
character  and  take  up  molecular  oxygen  witli  the  formation  of 
peroxides. 

(6)  The  Peroxydases. — These  are  non-albuminous  bodies 
which  contain  manganese  and  aluminium  and  in  some  instances 
iron  and  possibly  copper.  They  are  quite  stable  and  can  be  demon- 
strated by  means  of  hydrogen  peroxide,  which  they  decompose.  In 
themselves — /.  e.,  in  the  ab.sence  of  peroxides — they  are  incapable 
of  causing  oxidations,  but  they  increase  the  power  of  oxidation  of 
the  peroxides  very  materially. 

(c)  The  Katalases. — Tiiese  decompose  hydrogen  peroxide  kat- 
alytically  with  the  liberation  of  oxygen.  They  are  only  feeble 
oxidizing  agents. 

Conjointly  the  oxygenases  and  peroxydases  are  also  spoken  of  as 
oxydases.  Their  mode  of  action  consists  essentially  in  the  with- 
drawal of  two  atoms  of  hydrogen  with  the  formation  of  water,  and 
the  occasional  addition  of  one  atom  of  oxygen.  At  times,  however, 
a  more  eneriretic  oxidation  is  observed.  The  peroxydases  are  of 
supreme  biological  interest  owing  to  their  power  of  decomposing 
peroxides,  which,  according  to  Engler's  views,  form  the  starting- 
point  of  all  oxidations  in  the  animal  body. 

Aside  from  the  common  tests  for  oxydases,  viz.,  blueing  of  tincture 
of  guaiac  and  decomposition  of  hydrogen  peroxide,  ])henol]ihthalin 
may  also  be  employed ;  this  is  oxidized  to  jjhenolphthalein,  which 


CLASSIFICATION.  .  119 

may  then  be  estimated  colorimetrieally.  The  katalases,  in  contra- 
distinction to  the  oxydases  proper,  do  not  bhie  tinctures  of  guaiac 
either  directly  or  in  the  presence  of  hydrogen  peroxide. 

The  oxydases  occur  widely  distributed  in  the  animal  and  vegeta- 
ble world  ;  they  include  the  laccase,  which  causes  the  formation  of 
the  black  Japanese  lacquer ;  various  tyrosinases,  one  of  which  causes 
the  transformation  of  tyrosin  to  horaugentisinic  acid,  while  another 
is  responsible  for  the  formation  of  the  black  pigment  secreted  by 
the  octopus ;  an  indophenol  oxydase,  which  forms  indophenol  from 
paraphenylendiamin  and  rr-naphthol  ;  aldehydases  which  oxidize 
aldehydes  to  the  corresponding  acids,  etc. 

To  this  group  probably  also  belong  the  glucolytic  ferments,  which 
have  been  demonstrated  in  many  organs  of  the  animal  body,  notably 
the  muscle-tissue  and  the  liver;  in  both  instances  they  are  apparently 
activated  by  a  kinase  furnished  by  the  pancreas. 

12.  The  Coagulating  Ferments. — These  include  the  fibrin  ferment 
which  causes  the  coagulation  of  blood  ;  various  milk-curdling  fer- 
ments which  occur  both  in  the  animal  and  the  vegetable  world  ;  a 
ferment  which  is  thought  to  be  responsible  for  the  coagulation  of 
myosin  ;  pectase,  which  coagulates  the  pectins  of  plants  and  leads  to 
the  formation  of  pectic  acid,  etc. 

13.  Reducing  Ferments  (Reductases). — Such  ferments  apparently 
also  exist.  One  has  been  described  which  reduces  sulphur  to  hydro- 
gen sulphide. 


CHAPTER  VII. 

THE  DIGESTIVE  FLUIDS. 

As  I  have  pointed  out,  the  greater  portion  of  the  food-stuffs  which 
are  ingested  by  animals  cannot  be  utilized  as  such  directly,  but  must 
first  be  transformed  into  material  that  is  capable  of  diffusing  through 
animal  membranes.  These  changes  occur  in  the  gastro-intestinal 
tract,  and  are  effected  by  the'  secretions  of  the  various  digestive 
glands,  viz.,  the  saliva,  the  gastric  juice,  the  pancreatic  juice,  the 
succus  entericus,  and  the  bile. 

THE  SALIVA. 

General  Characteristics. — The  saliva  is  the  secretory  product 
of  the  salivary  glands,  viz.,  the  parotid,  the  submaxillary,  and  the 
sublingual  glands,  to  which  the  secretion  of  the  smaller  mucous 
glands  of  the  oral  cavity  is  further  added. 

The  saliva  is  a  colorless,  inodorous,  tasteless,  somewhat  stringy 
and  frothy,  opalescent  fluid,  which  normally  possesses  a  slightly  alka- 
line reaction  and  a  specific  gravity  ranging  between  1.002  and  1.008. 
A  slightly  acid  reaction  may,  however,  also  be  observed,  and  is  then 
referable  to  the  presence  of  lactic  acid,  which  is  formed  through  the 
activity  of  micro-organisms,  from  food-material  that  has  gathered 
between  the  teeth  or  from  desquamated  epithelial  cells.  For  this 
reason  also  we  find  an  acid  reaction  of  the  mouth-cavitv  on  risine: 
in  the  mornnio^. 

On  microscoi)ical  examination  the  saliva  is  seen  to  contain  a 
variable  number  of  pavement  epithelial  cells  and  so-called  salivary 
corpuscles.  These  are  identical  with  the  mucous  corpuscles,  which 
are  fijund  in  all  mucous  membranes,  and  represent  young  leucocytes 
that  have  not  entered  the  blood-current.  They  are  derived  from  the 
lymph-follicles  of  the  mucous  membrane,  and  in  the  case  of  the 
saliva,  no  doubt,  to  a  great  extent  from  the  tonsils.  In  addition 
we  find  innumerable  bacteria  and  at  times  also  schizomycetes  and 
moulds.  On  standing,  the  liquid  becomes  turbid,  owing  to  pre- 
cipitation of  calcium  carbonate,  which  frequently  also  forms  a  fine, 
iridescent  film  on  the  surface.  This  phenomenon  is  due  to  the 
escape  of  carbon  dioxide  from  the  saliva,  and  explains  the  formation 
of  tartar  on  the  teeth,  as  also  the  origin  of  the  somewhat  uncommon 
salivary  concretions  in  the  larger  ducts  of  the  glands. 

Amount. — The  amount  of  saliva  that  is  secreted  in  the  twenty- 
four  hours  varies  some^\"hat  even  in  health,  but  probably   does  not 

120 


THE  SALIVA.  121 

exceed  1500  c.c.  It  depends  upon  the  amount  of  nutriment  in- 
gested, the  act  of  chewing,  the  cliaracter  of  the  food,  the  mental 
condition,  etc.  Frig[it,  may  arrest  its  flow  entirely.  After  the 
administration  of  pilocarpin,  or  during  the  inhalation  of  ether,  an 
abundant  secretion  of  saliva  occurs,  and  it  is  thus  possible  to  col- 
lect in  the  human  being  sufficient  quantities  for  analysis.  Atropin 
acts  in  the  opposite  manner,  and  can  arrest  the  flow  entirely. 

To  a  certain  extent  the  amount  secreted  is  dependent  upon  the 
blood-pressure,  but  it  does  not  follow  that  the  saliva  results  from  the 
blood-j)hisma  through  a  simple  ])rocess  of  filtration.  We  find  tliat 
in  the  submaxillary  gland,  for  example,  the  secrc^tion  continues  for 
some  time  even  after  decapitation  of  the  animal.  The  secretory 
pressure,  moreover,  is  very  much  greater  than  the  blood-press- 
ure ;  and  after  the  administration  of  atropin,  which  paralyzes  the 
secretory  nerves,  we  further  find  that  while  electrical  stimulation 
of  the  chorda  calls  forth  an  increased  circulation  in  the  gland,  a 
secretion  of  saliva  does  not  occur.  These  experiments  show  that 
the  secretion  of  the  saliva  cannot  be  referable  to  a  simple  process 
of  filtration,  but  must  depend  upon  a  special  secretory  activity  on 
the  part  of  the  alveolar  cells.  We  thus  also  find  that  the  salivary 
glands  are  capable  of  eliminating  certain  chemical  substances,  such 
as  bromides  and  iodides,  from  the  body,  while  others,  like  iron  com- 
pounds, for  example,  are  not  removed  through  this  channel,  if  we 
disregard  the  trace  which  is  normally  present. 

Chemical  Composition. — The  chemical  composition  of  the  saliva 
is  qualitatively  fairly  constant.  Quantitative  variations,  howev^er, 
are  common.  This  is  to  a  certain  extent  owing  to  the  fact  that  the 
different  glands  are  not  all  of  one  kind.  In  the  human  being  the 
parotids  are  thus  albuminous  glands,  the  sublinguals  mucous  glands, 
while  the  submaxillary  glands  furnish  a  mixed  secretion.  The 
character  of  the  secretion,  moreover,  may  vary  with  one  and  the 
same  gland.  The  salivary  glands  all  have  a  double  nerve-supply, 
which  is  partly  of  cerebral  origin  and  partly  derived  from  the  sym- 
pathetic system,  and  as  the  one  or  the  other  set  of  fibres  exercises 
its  stimulating  effect,  the  composition  of  the  individual  secretions 
will  vary.  In  the  submaxillary  gland  of  the  dog,  in  which  these 
relations  have  been  especially  studied,  on  stimulation  of  the  sym- 
pathetic fibres  a  secretion  is  furnished  which  is  less  abundant,  but 
contains  a  larger  amount  of  solids,  than  the  secretion  obtained  on 
stimulation  of  the  chorda.  Tiiis  is  well  shown  in  the  following 
table,  which  is  taken  from  Kiihne : 

Sympathetic  saliva.  Chorda  saliva. 

Specific  gravity     .    .    .  1.007-1.018  1.004-1.00(5 

Solids 16-18  pro  mille  12-14  pro  niille 

On  dividing  all  the  nerves  which  supply  the  salivary  glands,  or 
following  the  administration  of  curare,  the  secretion  still  continues 
for  a  while,  but  tiie  saliva  which  is  thus  furnished  contains  scarcely 
any  solid  material,  and  is  termed  paralytic  saliva. 


122  THE  DIGESTIVE  FLUIDS. 

Qualitatively,  as  has  just  been  stated,  the  normal  mixed  saliva  is 
of  fairly  constant  composition.  The  quantitative  variations  M'hich 
may  occur  in  health  are  seen  from  the  following  analyses  of  human 
saliva,  which  are  taken  from  Hammarsten  : 

Frerichs.  Berzelius.        Hammerbacher. 

Water   .    .        994.1  992.9  994.2 

Solids 5.9  7.1  5.8 

Mucus  and  epitlieliuiu 2.13  1.3  2.2 

.Soluble  organic  matter 1.42  3.8  1.4 

Inorganic  salts 2.19  1.9  2.2 

Potassium  sulphocyanide  ....  0.10  .    .  0.04 

An  analysis  of  the  inorganic  salts,  moreover,  calculated  for  1000 
parts  by  weight  of  mineral  ash,  gave  the  folloM'ing  results  : 

Potassium 457.2 

Sodium 95.9 

Oxide  of  iron 50.11 

Oxide  of  magnesium      1.55 

Sulphuric  acid  (as  SO3) 63.8 

Phosphoric  acid  (as  P2O5) 188.48 

Chlorine 183.52 

The  albumins  proper  of  the  saliva  are  said  to  be  similar  to  those 
of  the  blood-serum,  but  are  present  in  only  very  small  amount. 
They  may,  in  fact,  be  regarded  as  accidental  constituents,  as  the 
greater  portion  of  the  albumins  which  enter  into  the  composition  of 
the  glandular  cells  is  no  doubt  transformed  into  the  specific  secretory 
products  of  these  glands,  viz.,  into  mucin  and  the  amylolytic  fer- 
ment ptyalin.  In  the  cells  proper,  however,  these  substances  aj)par- 
ently  do  not  exist  as  such,  but  as  mucinogen  and  ptyalingen,  Avhich 
are  later  transformed  into  mucin  and  ptyalin,  respectively.  As  a 
matter  of  fact,  it  is  possible  to  obtain  the  inactive  ptyalinogen  from 
the  solids  of  the  horse,  and  to  transform  it  artificially  into  the  active 
ferment.  To  this  end,  it  is  only  necessary  to  collect  the  saliva  from 
the  parotid  gland  of  the  horse  under  antiseptic  precautions,  and  to 
prevent  the  further  access  of  micro-organisms.  A  secretion  is  thus 
obtained  which  is  perfectly  inert  when  brought  in  contact  with 
starch  solution,  while  a  corresponding  specimen  that  has  been  ex- 
posed to  the  air  at  once  begins  to  manifest  the  specific  activity  of 
free  ptyalin.  Of  the  manner  in  which  this  transformation  is  eifected 
in  the  mouth,  we  are  as  yet  ignorant,  but  it  appears  that  the  bacteria 
which  are  here  normally  present  are  of  importance.  Similar  results 
are  reached  when  the  finely  hashed  glands  are  extracted  with  chloro- 
form-water, until  the  active  ferment  can  no  longer  be  obtained  in 
this  manner.  On  subsequent  treatment  with  a  very  dilute  solution 
of  acetic  acid  other  extracts  can  then  l)e()l)tained,  which  are  as  active 
as  the  first,  thus  showing  that  a  substance  must  have  been  present 
which  could  not  be  isolated  with  the  chloroform-water,  but  which 
can  be  transformed  into  ptyalin  by  means  of  acetic  acid. 

Ptyalin. — The  ptyalin  or  salivary  diastase,  as  it*  is  also  termed,  is 
an  amylolytic  ferment,  and  as  such  capable  of  causing  the  inversion  of 


THE  SALIVA.  123 

starch  to  sugar.  This  can  be  readily  demonstrated  as  follows  :  A  few 
cubic  centimeters  of  saliva  are  added  to  a  small  amount  of  starch  solu- 
tion and  kept  at  a  temperature  of  about  35°  C.  If  a  drop  of  this  mixt- 
ure is  then  tested  at  intervals  of  about  one  minute  with  a  dilute  solu- 
tion of  iodine,  it  will  be  observed  that  the  blue  color,  which  is  first 
obtained  by  bringing  a  drop  of  the  two  solutions  together,  soon  gives 
place  to  a  violet,  and  then  to  a  mahogany  brown,  and  that  still 
later  no  color-reaction  whatever  occurs.  As  soon  as  this  point  is 
reached  a  small  amouut  of  the  starch  mixture  is  examined  with 
Trommer's  or  Fehling's  solution  (^see  page  299),  when  the  presence 
of  sugar  can  be  established.  The  sugar  which  thus  results  is  maltose, 
while  the  intermediary  products  which  are  formed  during  the  inver- 
sion of  the  starch  are  represented  by  erythrodextrin,  achroodextrin, 
and  isomaltose.  The  reaction  w'hich  takes  place  may  be  represented 
by  the  equations : 

(1)  (C„H,Ao)5i  +    3H,0  =  3[(C,,H,oO,o)„-C,,H,.Ai] 

Amidulin.  Erythrodextrin. 

(2)  3[(C„H,oO,o)n.C,,K,,0,i]  +    6H,0  =  9[(Ci,H,oOio)5.C,,H,,0,i] 

Achroodextrin. 

(3)  9[(Ci,H,oO,o)5.C,,H,,OiJ    -  45H.,0  =  UC,,YL,,0,,  =  54C„H,20„ 

Isomaltose.  Maltose. 

We  shall  return  to  these  reactions  in  the  next  chapter,  when  the 
digestion  of  the  food  is  considered  in  detail. 

To  isolate  the  ptyalin  from  saliva,  the  following  method,  which 
has  been  suggested  by  Gautier,  may  be  employed  :  To  a  large 
quantity  of  saliva  98  per  cent,  alcohol  is  added  so  long  as  a  floccu- 
lent  precipitate  is  seen  to  form.  This  is  collected  on  a  small  filter 
and  dissolved  with  a  small  amount  of  distilled  water.  The  solu- 
tion is  treated  with  a  few  drops  of  a  solution  of  bichloride  of  mercury, 
in  order  to  remove  any  albuminous  material  that  may  be  present. 
In  the  filtrate  the  excess  of  the  bichloride  is  removed  with  hvdrogen 
sulphide,  when  the  remaining  liquid  is  evaporated  to  dryness  at  a 
temperature  not  exceeding  40°  C,  and  the  residue  is  treated  with 
strong  alcohol.  The  insoluble  portion  is  then  dis.solved  with  a  small 
amouut  of  distilled  water,  filtered,  dialyzed  in  order  to  remove  inor- 
ganic salts,  and  finally  precipitated  with  absolute  alcohol,  w^hen  the 
ptyalin  will  separate  out  in  light  flakes.  Obtained  in  this  manner, 
ptyalin  is  a  white  amorphous  substance,  which  is  solulile  in  water, 
dilute  alcohol,  and  glycerin.  In  neutral  or  slightly  alkaline  solu- 
tion, but  not  in  acid  solution,  it  rapidly  transforms  boiled  starch 
into  sugar  at  a  temperature  of  from  36°  to  40°  C.  Heated  to  a 
temperature  of  60°  C,  its  solutions  lose  this  power,  and  it  is  thus 
possible  to  distinguish  between  ptyalin  and  the  diastatic  ferment  of 
vegetable  oris^in,  for  which  the  optimum  temperature  lies  between 
60°  aiul  65°  C. 

Of  special  interest  is  the  fact  that  the  transformation  of  starch 
into  sugar  ceases  as  soon  as  the  latter  is  present  to  the  extent  of 
from  2  to   2.5  per  cent.     This  phenomenon  is  common  to  all  enzy- 


124  THE  DIGESTIVE  FLUIDS. 

miitie  processes,  and  is  probably  referable  to  the  establishment  of  a 
certain  equilibrium  of  reaction,  A  complete  transformation  of  the 
starch  could  occur  only  if  the  resulting  sugar  were  removed  as 
rapidly  as  it  is  formed.  So  long  as  it  is  present,  the  reversible 
action  of  the  enzyme  becomes  manifest,  and,  analogous  to  the  rever- 
sion of  glucose  to  maltose,  a  similar  retransformation  of  maltose  to 
dextrin  no  doubt  occurs. 

The  amount  of  ptyalin  which  is  secreted  in  the  twentv-four 
hours  has  not  been  determined.  Its  activity,  as  would  be  ex- 
pected, is  subject  to  considerable  variations.  It  is  greatest  in  the 
morning  on  rising,  and  then  steadily  diminishes  during  the  day. 
Immediately  before  meals,  however,  a  temporary  increase  is  ob- 
served, which  is  then  followed  by  a  marked  decrease. 

Of  the  chemical  nature  of  ptyalin  but  little  is  known.  Like  all 
other  ferments,  it  is  generally  regarded  as  an  albuminous  substance, 
and  on  the  application  of  dry  heat  it  develops  the  characteristic  odor 
of  burning  albumins.  It  is  nitrogenous,  but  does  not  give  the 
xanthoproteic  reaction.  From  its  solutions  it  can  be  precipitated 
with  acetate  and  subacetate  of  lead,  while  bichloride  of  mercury  and 
the  salts  of  platinum,  as  also  tannic  acid,  are  without  eft'ect. 

In  the  human  l)eing  ptyalin  is  formed  in  the  parotid  and  the 
submaxillary  glands  and,  as  will  be  seen  later,  also  in  the  pancreas, 
while  the  sublingual  glands  apparently  yield  no  ptyalin.  In  other 
animals  its  presence  in  the  saliva  is  variable.  In  the  typical 
carnivora  it  is  said  to  be  absent,  while  in  the  saliva  of  all  herbivor- 
ous animals  it  is  uniformly  found. 

Of  other  ferments,  human  saliva  apparently  also  contains  traces 
of  maltase,  and  of  an  oxydase  of  unknown  character ;  invertin, 
however,  has  not  been  found.  Consequently  an  inversion  of  maltose 
to  glucose  may  also  take  place  in  the  early  stages  of  carbohydrate 
digestion,  but  is  certainly  insignificant  in  extent. 

The  (Vigc^tive  imjiortance  of  the  saliva  is,  in  man,  at  least,  but 
slight,  as  ptyalin  is  rapidly  destroyed  by  contact  with  the  acid 
gastric  juice.  During  the  process  of  mastication  and  deglutition, 
moreover,  it  has  scarcely  time  to  eflPect  much  change,  and  in  experi- 
ments in  vitro  we  find  that  the  amount  of  maltose  which  is  formed 
by  the  saliva  from  starch  is  small.  The  im])ortance  of  the  salivary 
glands  as  digestive  glands  has  thus  been  much  overrated,  and  it  has 
been  conclusively  demonstrated  that  their  function  has  mostly  to  do 
with  the  pre^iaration  of  the  food  for  the  act  of  deglutition.  This  is, 
of  course,  greatly  facilitated  by  its  thorough  lubrication  with  the 
mucus  that  is  furnished  by  the  salivary  glands,  and  which  in  reality 
represents  the  most  important  constituent  of  the  saliva. 

Mucin. — The  mucin  of  the  saliva  is  derived  from  the  submaxil- 
lary and  sublingual  glands,  as  also  from  the  small  mucous  glands 
which  are  found  imbedded  in  the  mucous  membrane  of  the  mouth. 
Its  formation  in  the  salivary  glands  is  apparently  under  the  control 
of  the  sympathetic  nervous  system,  as  it  is  secreted  in  much  larger 


THE  SALIVA.  125 

amounts  on  stimulation  of  these  fibres  than  of  the  corresponding 
cerebral  fibres.  Accordino;  to  Levene,  the  submaxillary  mucin  con- 
tains the  chondroitin-sulphuric  acid  complex,  or  a  closely  allied 
group. 

To  the  presence  of  the  mucin  the  viscid,  stringy  character  of  the 
saliva  is  due.  The  substance  can  be  obtained  by  precipitation  with 
acetic  acid,  and  it  is  to  be  noted  that,  in  contradistinction  to  the 
mucin-like  substances  which  belong  to  the  class  of  the  nucleo-albu- 
mins,  tlie  precipitated  mucin  is  insoluble  in  an  excess  of  the  acid. 
In  dilute  solutions  of  the  alkalies  it  is  soluble,  and  it  is  thus 
possible  by  repeated  precipitation  and  solution  to  obtain  the  mucin 
in  fairly  pure  form.  In  alcohol  and  water  it  is  insoluble,  although 
in  the  latter  it  swells  to  form  a  jelly-like  material.  Unlike  the 
albumins,  it  is  not  coagulated  by  heat;  but,  like  these,  it  gives  the 
xanthoproteic  reaction,  the  biuret  reaction,  and  Millon's  reaction. 
It  contains  also  a  small  amount  of  sulphur.  On  boiling  with  dilute 
mineral  acids  mucin  is  decomposed  into  a  substance  which  resembles 
acid  albumin,  and  into  a  carbohydrate-like  body  which  reduces 
Fehling's  solution.  This  has  been  shown  to  be  a  glucosamin. 
According  to  Miiller,  it  is  present  to  the  extent  of  23.5  per  cent. 
On  decomposition  w'xih  strong  mineral  acids  mucin  yields  leucin, 
tyrosin,  and  Itevulinic  acid  (see  also  page  68).  In  the  dry  state  it 
occurs  as  a  white  or  yellowish-gray  powder. 

Within  the  cells  mucin  exists  as  so-called  mucinogen,  which  prob- 
ably represents  a  compound  of  mucin  with  an  additional  albuminous 
substance. 

Sulphocyanides. — Traces  of  sodium  sulphocyanide  are  in  man 
usually  found  in  every  specimen  of  normal  saliva.  It  is  secreted 
by  all  the  salivary  glands,  but  in  largest  amount  by  the  parotids. 
In  other  animals  its  presence  is  not  so  constant,  and  in  some  indeed 
it  is  not  found.     In  man  also  it  is  at  times  absent. 

To  demonstrate  the  presence  of  sulphocyanides,  it  usually  suffices 
to  treat  a  few  cubic  centimeters  of  saliva,  which  have  been  slightly 
acidified  with  hydrochloric  acid,  with  a  few  dro])s  of  a  very  dilute 
solution  of  perchloride  of  iron,  when  a  red  color  will  be  seen  to 
develop.  If  no  result  is  obtained  in  this  manner,  a  larger  quantity, 
such  as  100  c.c.  is  evaporated  to  a  small  volume  and  tested  as 
described. 

Nitrites. — Small  amounts  of  nitrites  may  also  at  times  be 
observed,  and  are  no  doubt  derived  from  the  nitrates  ingested.  To 
test  for  these,  about  10  c.c.  of  saliva  are  treated  with  a  few  drops 
of  Ilasvay's  reagent,  and  heated  to  a  temperature  of  80°  C,  wdien 
in  the  presence  of  nitrites  a  red  color  develops. 

Ilasvay's  reagent  is  prepared  as  follows  :  0.5  gramme  of  sulph- 
anilic  acid  in  150  c.c.  of  dilute  acetic  acid  is  treated  with  0.1 
granime  of  naphtylamin  dissolved  in  20  c.c.  of  boiling  water.  After 
standing  for  some  time  the  supernatant  fluid  is  poured  off,  and  the 


126  THE  DIGESTIVE  FLUIDS. 

sediment  dissolved  in  150  c.o.  of  dilute  acetic  acid.  The  solution 
is  kept  in  a  sealed  bottle. 

Extractives. — Of  extractives,  normal  saliva  contains  a  small 
amount  of  urea,  and  traces  of  cholesterin,  lecithin,  and  leucin.  In 
gouty  conditions  uric  acid  has  been  found  ;  sugar,  the  biliary  pig- 
ments, and  biliary  acids  are  not  eliminated  through  the  saliva. 

Mineral  Constituents. — The  mineral  constituents  of  saliva  con- 
sist to  the  extent  of  90  to  92  per  cent,  of  soluble  salts,  among  which 
the  chlorides  greatly  predominate,  and  of  about  6  per  cent,  of  salts, 
which  are  principally  represented  by  the  carbonates  and  phosphates 
of  calcium  and  magnesium,  which  are  held  in  solution  by  the  free 
carbonic  acid  of  the  saliva.  In  addition,  a  trace  of  iron  is  found. 
Following  the  administration  of  bromides  and  iodides  a  notable 
elimination  of  these  salts  occurs  through  this  channel. 

Gases. — Of  gases,  which  are  present  in  a  state  of  solution,  we 
find  about  20  c.c.  for  every  100  grammes  of  saliva.  Of  these,  19 
c.c.  are  represented  by  carbon  dioxide,  while  oxygen  and  nitrogen 
together  amount  to  only  1  c.c. 

THE   GASTRIC  JUICE. 

General  Considerations. — The  gastric  juice  is  the  secretory 
product  of  the  glandular  structures  of  the  stomach,  and  the  only 
digestive  fluid  which  presents  an  acid  reaction.  In  jjwre  form  it  is 
best  obtained  from  animals  after  ligating  the  ducts  of  the  salivary 
glands  and  establishing  a  fistulous  c»pening  on  the  outer  abdominal 
walls.  If  the  mucous  membrane  is  then  ap])ropriately  stimulated, 
a  clear  or  but  slightly  opalescent  yellowish  fluid  is  obtained,  which 
has  a  very  characteristic  odor  and  a  strongly  acid  reaction.  Its 
density  varies  between  1.001  and  1.010. 

On  microscopic  examination  are  found  epithelial  cells  from  the 
lining  of  the  glandular  ducts,  goblet-cells,  mucous  corpuscles,  free 
nuclei,  and  a  variable  number  of  bacteria.  In  addition,  we  often 
observe  small  tapioca-like  bodies,  which  under  the  microscope  are 
seen  to  contain  numerous  formations  resembling  snail-shells,  and 
which  probably  consist  of  altered  mucin. 

Amount. — Of  the  total  amount  of  gastric  juice  secreted  in  the 
twenty-four  hours,  but  little  is  known.  Its  secretion  is  influenced  by 
numerous  factors,  such  as  the  appetite,  the  quality  and  quantity  of 
the  food  ingested,  the  age  and  sex  of  the  individual,  the  time  of  day 
(notably  in  relation  to  the  taking  of  food),  the  various  emotions,  etc. 
According  to  Bidder  and  Schmidt,  the  amount  corresponds  to  about 
one-tenth  of  the  body-M(Mght,  so  that  a  man  weighing  70  kilo- 
grammes would  secrete  about  7000  grammes  in  the  twenty-four 
hours.  This  figure,  however,  I  regard  as  too  high,  and  am  inclined 
to  place  the  amount  at  from  2000  to  3000  c.c. 

The  non-disresting:  stomach  of  the  doer  and  other  animals   is  said 


THE  GASTRIC  JUICE.  127 

to  contain  no  flnid ;  in  man,  however,  a  small  amount  of  gastric 
juice  can  usually  be  obtained  by  means  of  the  stomach-tube,  vary- 
ing between  1  and  60  c.c.  Larger  amounts  may  be  found  under 
pathologic  conditions,  and  in  the  so-called  Magensaftfluss  of  the 
Germans  it  is  not  rare  to  find  as  much  as  1000  c.c.  in  the  early 
morning,  before  any  food  has  been  taken. 

The  amount  of  fluid  which  can  be  normally  obtained  from  the 
digesting  organ  is  likewise  variable.  It  depends  upon  the  amount 
of  liquid  ingested,  the  period  of  digestion,  the  character  of  the  food, 
the  size  and  motor  power  of  the  stomach,  etc.  Exact  figures,  how^- 
ever,  are  lacking  to  represent  these  relations,  and  it  is  manifest  that 
such  figures  must  always  have  reference  to  more  or  less  diluted 
gastric  juice. 

Chemical  Composition. — A  general  idea  of  the  chemical  com- 
position of  the  gastric  juice  may  be  formed  from  the  following  analy- 
ses, which  are  taken  from  C.  Schmidt ;  but  it  is  to  be  noted  that 
the  specimen  of  human  gastric  juice  was  contaminated  with  saliva 
and  somewhat  diluted  with  water  (the  figures  have  reference  to  1000 
parts) : 

Human  gastric  juice,  Gastric  juice  of 

containing  saliva  dog,  free 

with  water.  from  saliva. 

Water 994.40 973.0 

Solids 5.60  .......  27.0 

Organic  material 3.10 17.1 

Mineral  salts 2.19 6.7 

Sodium  cldoride 1.46 2.5 

Calcium  chloride      0.06 0.6 

Potassium  chloride 0.55 1.1 

Ammonium  chloride 0.5 

Calcium  phosphate 1.7 

Magnesium  phosphate 0.2 

Iron 0.12 0.1 

Free  hydrochloric  acid 0.20 0.1 

Acidity  of  the  Gastric  Juice. — It  has  now  been  definitely 
established  that  the  acidity  of  normal  gastric  juice  is  referable  to 
the  presence  of  free  hydrochloric  acid,  and  to  this  only.  This  can 
be  shown  by  estimating  the  amount  of  chlorine  and  all  basic  sub- 
stances that  are  present,  M'hen  it  will  be  found  that  after  the  acid 
affinities  of  the  latter  have  been  saturated,  a  certain  amount  of 
chlorine  still  remains,  which  can  be  referable  only  to  hydrochloric 
acid,  and  corresponds  in  its  degree  of  acidity  to  that  observed  in 
the  gastric  juice  itself. 

During  the  process  of  digestion,  however,  other  factors  enter  into 
consideration.  In  the  beginning  of  digestion  lactic  acid  is  alwavs 
present  when  carbohydrates  form  part  of  the  meal.  Its  amount, 
however,  is  then  quite  small,  and  after  the  ingestion  of  Ewald's 
test-breakfast,  for  example,  does  not  exceed  0.3  ]iro  mille.  The 
occurrence  of  larger  quantities  of  lactic  acid,  as  from  1  to  3  ]>ro 
mille,  is  always  abnormal,  and  in  many  cases  indicative  of  the 
exi.stence  of  carcinoma  of   the  stomach.     Durino-  the  later  stashes 


128  THE  DIGESTIVE  FLUIDS. 

of  digestion,  Mhen  hydrochloric  acid  is  found  in  a  free  state,  lactic 
acid  disappears.  Its  origin,  under  normal  conditions  at  least,  is 
referable  to  the  action  of  certain  bacteria,  such  as  the  Bacterium 
lactis,  the  Bacillus  lactis  aerogenes,  etc.,  upon  starches  and  sugars, 
as  represented  by  the  equations  : 

(1)  2QH,o05  -  HP  =  C,,JI,,0„ 

Starch.  Lactose. 

(2)  C,2H,2C„  -T-  H2O  =  4C,H,;03 

Lactose.  Lactic  acid. 

Other  organic  acids,  such  as  butyric  acid  and  acetic  acid,  are  usu- 
ally not  found  in  the  gastric  contents  unless  large  amounts  of  milk, 
carbohydrates,  or  alcohol  have  been  ingested.  In  such  event,  how- 
ever, they  may  be  present,  and,  like  lactic  acid,  are  then  referable  to 
the  action  of  certain  micro-organisms.  They  are  essentially  of  patho- 
logical significance. 

It  is  thus  seen  that  even  during  the  process  of  digestion  the 
acidity  of  the  gastric  contents  is,  under  normal  conditions,  scarcely 
influenced  by  acids  other  than  hydrochloric  acid.  It  should 
be  noted,  however,  that  following  the  ingestion  of  food  hydro- 
chloric acid  does  not  appear  in  a  free  state  at  once,  but  only 
after  the  affinities  of  the  albuminous  constituents  of  the  food  have 
been  saturated.  A^'e  consequently  find  that  while  in  the  begin- 
ning of  digestion  the  acidity  of  the  stomach-contents  is  largely 
referable  to  such  combined  acid,  in  the  later  phases  of  digestion  two 
factors  enter  into  consideration,  viz.,  free  and  combined  hydrochloric 
acid.  The  period  at  which  free  acid  appears  as  such  varies,  of 
course,  with  the  character  of  the  meal,  anrl  directly  with  the  amount 
of  proteids  ingested.  After  the  administration  of  Ewald's  test- 
breakfast  free  acid  is  thus  found  only  at  the  expiration  of  about 
thirty-five  minutes  ;  while  after  the  administration  of  Riegel's  test- 
dinner,  which  contains  much  larger  amounts  of  albumin,  two  hours 
must  elapse  before  free  acid  can  be  demonstrated. 

Acid  salts,  finally,  play  only  a  small  part  in  determining  the  total 
acidity  of  the  gastric  juice  ;  and  it  is  thus  clear  that  unless  carbo- 
hydrates, much  fat,  and  alcohol  have  been  ingested,  hydrochloric 
acid,  either  in  a  free  state  or  in  combination  with  all)umin,  or  both, 
is  the  sole  factor  which  enters  into  consideration.  Under  patholr)gical 
conditions,  on  the  other  hand,  lactic  acid,  butyric  acid,  and  acetic 
acid  may  also  play  a  part ;  but  then  hydrochloric  acid  is  usually  not 
present,  and  the  acidity  of  the  gastric  contents  is  hence  largely 
referable  to  fermentative  changes  which  have  taken  place  in  the 
stomach. 

Determination  of  the  Total  Acidity  of  the  Gastric  Contents. 
— Five  or  10  c.c.  of  the  filtered  gastric  contents  are  titrated  with  a 
decinormal  solution  of  sodium  hydrate,  using  phoiiolphthalein  as  an 
indicator,  until  the  rose  color,  wJiich  appears  on  the  addition  of  each 
drop  of  the  sodium  hydrate  solution,  no  longer  disappears  on  stirring 
or  is  intensified  by  the  addition  of  a  further  drop.     The  number  of 


THE  GASTRIC  JUICE.  129 

cubic  centimeters  employed  to  bring  about  this  reaction,  multiplied 
by  0.00365,  indicates  the  acidity  of  the  5  or  10  c.c.  of  gastric  juice 
in  terms  of  hydrochloric  acid. 

Amount. — The  degree  of  acidity  of  the  gastric  juice  is  usually 
fairly  constant,  and  in  man  varies  between  0.15  and  0.25  per  cent. 
It  is  influenced  to  a  certain  extent  by  the  character  of  the  food ;  for 
instance,  following  the  administration  of  a  meal  rich  in  proteids, 
somewhat  larger  amounts  are  obtained  than  after  the  ingestion  of 
carbohydrates  or  fats.  The  smallest  amounts  are  found  after  the 
ingestion  of  water.  After  Ewald's  test-breakfast,  which  consists  of 
from  35  to  70  grammes  of  ^vheat  bread  and  300  to  400  c.c.  of 
water,  or  weak  tea  without  sugar,  the  maximum  acidity  is  reached 
in  about  one  hour,  and  corresponds  to  1.5  to  1.75  pro  mille.  Follow- 
ing tlie  ingestion  of  lliegel's  test-meal,  on  the  other  hand,  which 
consists  of  a  plate  of  soup  (400  c.c),  200  grammes  of  beefsteak,  50 
grammes  of  wheat  bread,  and  200  c.c.  of  water,  the  amount  of 
hydrochloric  acid  increases  to  2.7  pro  mille,  after  from  one  hundred 
and  eighty  to  two  liundred  and  ten  minutes.  In  disease  still  higher 
figures  (5  p.  m.)  may  be  observed  ;  or  its  secretion  may  diminish 
below  the  normal,  and  may  even  cease  altogether. 

Hydrochloric  Acid. — Origin. — Tlie  hydrochloric  acid  of  the 
gastric  juice  is  furnished  by  the  so-called  parietal,  adelomorphous,  or 
oxyntic  cells,  which  are  principally  found  in  the  glands  of  the 
fundus.  This  can  be  demonstrated  by  resecting  the  fundus,  then 
closing  one  end  with  a  fine  suture,  and  sewing  the  other  into  the 
abdominal  wound,  while  the  cardiac  portion  of  the  stomach  is 
joined  to  the  pyloric  end.  If  food  be  now  administered  to  the 
animal,  a  fluid  will  be  secreted  by  the  isolated  fundus  in  which  the 
presence  of  free  hydrochloric  acid  can  easily  be  shown.  If,  on  the 
other  hand,  the  pyloric  end  of  the  stomach,  in  which  no  parietal 
cells  are  found,  is  similarly  isolated,  no  acid  is  obtained,  but,  instead, 
a  strongly  alkaline  mucus. 

While  it  is  thus  clear  that  the  hydrocldoric  acid  is  furnished  by 
the  parietal  cells,  we  are  as  yet  ignorant  of  the  mechanism  by  which 
this  is  accomplished.  A  free  acid  is  manifestly  not  present  in  these 
cells,  as  can  be  shown  by  testing  with  litmus-pa]5er,  or  still  better  by 
injecting  potassium  ferrocyanide  and  lactate  of  iron  into  the  circula- 
tion of  an  animal,  when  it  will  be  observed  tliat  Berlin-blue  is 
formed  in  the  stomacli-cavity,  while  the  cells  themselves  remain 
unstained.  It  thus  follows  that  a  substance  must  either  be  present 
in  the  cells  which  is  capable  of  yielding  hydrochloric  acid  when 
secreted  to  the  outside,  or  a  mechanism  must  exist  by  which  the 
hydrochloric  acid,  though  formed  within  the  cells,  is  at  once  elim- 
inated. The  latter  view  is  now  generally  held.  That  the  hydro- 
chloric acid  is  derived  from  the  chlorides  of  the  blood  can  be 
regarded  as  an  established  fact.  It  may  thus  be  secreted  even 
though  no  food-stuffs  have  been  ingested  ;  and  Kahn,  moreover,  has 
shown  that  animals  in  which  the  chlorides  of  the  body  have  been 

9 


130  THE  DIGESTIVE  FLUIDS. 

artificially  reduced  to  that  minimum  which  is  tenaciously  retained 
are  no  longer  capable  of  secreting  hydrochloric  acid.  The  mech- 
anism, however,  by  which  the  formation  of  hydrochloric  acid  takes 
place  is,  as  has  been  stated,  unknown.  Ludwig  formerly  thought 
that  it  resulted  through  electrolytic  influences  within  the  cells,  and 
that  the  acid  then  diffused  out  into  the  lumen  of  the  glandular  duct 
before  the  alkaline  elements  of  the  cell  could  bring  about  its  neutral- 
ization. This,  however,  is  improbable,  and  the  view  that  is  now 
held  by  most  observers  is  that  expressed  by  Maly,  according  to 
which  the  hydrochloric  acid  results  from  a  mass-action  on  the  })art 
of  the  carbonic  acid  of  the  blood  upon  the  chlorides  in  the  body  of 
cells,  and  is  immediately  eliminated  to  the  outside,  while  the  result- 
ing bicarbonate  is  returned  to  the  blood.  We  know  that  within  the 
cells  carbon  dioxide  is  present  under  great  pressure.  Schlicrbcck 
thus  found  that  water  which  is  introduced  into  the  stomach  of  living 
dogs  after  a  variable  length  of  time  contains  a  certain  amount  of 
carbon  dioxide,  and  that  its  tension  rises  from  30  to  40  Hgmm. 
while  fasting,  to  130  to  140  Hgmm.  during  the  process  of  active 
digestion. 

Of  late,  Liebermann  has  further  suggested  that  lecithall)umin  may 
be  ])resent  in  the  parietal  cells  in  combination  Avith  sodium  chloride, 
and  that  hydrochloric  acid  may  result  from  this  through  a  mass- 
action  on  the  part  of  carbonic  acid,  which,  in  turn,  is  formed  within 
the  cells  as  a  result  of  an  increase  of  their  functional  activity. 

Significance  of  the  Hydrochloric  Acid. — The  assumption  that  the 
principal  function  of  the  hydrochloric  acid  consists  in  its  jiower  to 
render  the  pepsin  of  the  gastric  juice  physiologically  active,  viz., 
capable  of  bringing  about  the  transformation  of  albumins  into  albu- 
moses  and  peptones,  has  now  been  largely  abandoned.  We  know 
that  life  can  go  on  in  the  entire  absence  of  the  stomach,  as  has  been 
proved  not  only  by  ex]3eriments  on  animals,  but  also  by  operations 
wdiich  have  been  performed  on  the  human  being.  A  dog  whose 
stomach  was  almost  entirely  removed  by  Czerny  in  1876,  lived  for 
more  than  six  years  after  the  operation,  when  it  was  killed  in  Lud- 
wig's  laboratory ;  and  it  is  reported  that  the  animal  was  normal  in 
every  respect,  and  had  increased  in  weight  from  5850  grammes  to 
7000  g-rammes.  It  is  thus  manifest  that  while  the  hvdrochloric  acid 
of  the  gastric  juice  no  doubt  aids  in  the  process  of  albuminous  di- 
gestion, its  presence  to  this  end  is  not  imjicrativc,  and  the  question 
naturally  suggests  itself,  whether  the  secretion  of  such  large  amounts 
of  acid  does  not  serve  another  and  perhaps  more  important  })urpose. 
This  purpose  is  now  thought  to  bo  the  jirevention  of  ])utref;ictive 
changes  in  the  contents  of  the  stomach,  and  we  find,  as  a  matter  of 
fact,  that  albuminous  material  that  has  been  removed  from  the 
stomach  at  the  height  of  digestion  can  be  preserved  for  a  long  time 
without  undergoing  decomposition.  It  has  been  noted,  moreover, 
that  the  gastric  juice  is  also  capable  of  arrestinjs^  putrefactive  proc- 
esses when  these  have  becrun  before  the  ingestion  of  such  material. 


THE  GASTRIC  JUICE.  131 

This  problem  has  been  carefully  investigated,  and  we  now  know 
that  the  amount  of"  iiydi-ochloric  acid  which  is  present  at  the  height 
of  digestion  is  sufficient  at  least  to  arrest  the  activity  of  most 
bacteria  which  are  usually  ingested  together  with  food.  Some  of 
these,  however,  are  more  resistant  than  others,  and  among  them  the 
lactic  acid  bacteria  are  especially  stable.  We  can  accordingly  under- 
stand why  in  the  beginning  of  the  process  of  digestion  traces  of 
lactic  acid  are  usually  found.  So  soon,  however,  as  the  percen- 
tage of  hydrochloric  acid  has  increased  to  0.7  pro  mille  the  activity 
of  these  organisms  also  ceases.  Whether  or  not  the  gastric  juice 
actually  destroys  all  of  the  more  common  micro-organisms  that 
are  swallowed  has  not  been  determined,  but  there  is  evidence  to 
show  that  in  some  instances  at  least  the  spores  remain  unaiFected 
and  can  later  develop  in  the  alkaline  contents  of  the  small  intes- 
tine. In  this  manner  we  can  account  for  the  presence  of  the 
innumerable  bacteria  which  are  found  in  the  lower  intestinal 
tract.  It  must  be  remembered,  moreover,  that  in  the  beginning 
of  digestion — i.  e.,  when  free  hydrochloric  acid  is  as  yet  not  pres- 
ent— large  numbers  of  bacteria  may  also  pass  through  the  stomach 
unaffected,  as  combined  hydrochloric  acid  possesses  no  anti-fermenta- 
tive properties.  Some  of  the  more  important  pathogenic  bacteria 
are  unfortunately  more  resistant  than  the  common  benign  forms. 
This  is  true  especially  of  the  tubercle  bacillus,  and  in  many  cases  of 
the  anthrax  bacillus  and  some  of  the  pus  organisms,  while  the 
cholera  bacillus,  on  the  other  hand,  is  readily  destroyed. 

Tests  for  Free  Hydrochloric  Acid. — A  large  number  of  tests  have 
been  devised  for  tlie  ]>urpose  of  demonstrating  the  presence  of  free 
hydrochloric  acid  in  the  stomach-contents.  At  this  place  only  the 
more  important  ones  will  be  described,  which  are  employed  in  the 
clinical  laboratory. 

Topfer's  Test. — A  small  amount  of  the  filtered  gastric  contents 
is  treated  with  a  few  drops  of  a  0.5  per  cent,  alcoholic  solution  of 
dimethyl-amido-azobenzol,  when  in  the  presence  of  free  hydro- 
chloric acid  a  beautiful  cherry-red  color  develojjs  at  once,  which 
varies  in  intensity  with  the  amount  of  the  free  acid  present.  Com- 
bined hydrochloric  acid,  as  well  as  acid  salts  and  organic  acids,  in 
the  concentration  in  which  they  may  be  met  with  in  the  stomach- 
contents,  does  not  produce  this  color. 

The  delicacy  of  the  reagent  is  such  that  the  normal  yellow  color 
of  the  indicator  is  changed  to  a  reddish  tinge  upon  the  addition  of 
but  one  drop  of  a  -^-^  normal  solution  of  hydrochloric  acid  in  5  c.c. 
of  distilled  water,  viz.,  0.7  per  cent. 

Gunzburg's  Test. — The  reagent  consists  of  2  grammes  of  phloro- 
glucin  and  1  gramme  of  vanillin,  dissolved  in  100  grammes  of 
80  per  cent,  alcohol.  It  should  be  kept  in  a  dark-colored,  glass- 
stoppei'ed  bottle. 

A  few  drops  of  the  filtered  gastric  contents  are  carefully  evapo- 
rated with  an  equal  amount  of  the  reagent  on  a  plate  of  thin  porce- 


]P,2  THE  DIGESTIVE  ELl'IDS. 

lain  or  glass,  when  in  the  presence  of  free  hydrochloric  acid  a 
rose-colored  mirror  is  obtained,  which  varies  in  inten.-itv  Avitli  the 
amount  of  the  acid. 

Organic  acids  do  not  produce  the  reaction. 

The  delicacy  of  the  test  is  such  that  the  presence  of  0.05  gramme 
of  hydrochloric  acid  in  100  parts  of  water  can  be  demonstrated. 

Boas'  Test. — The  reagent  consists  of  5  grammes  of  resublimed 
resorcin  and  3  grammes  of  cane-sugar,  dissolved  in  100  grammes 
of  94  per  cent,  alcohol.  The  test  is  conducted  like  that  of  Giinz- 
bnrg,  but  it  is  necessary  to  heat  a  little  more  stronglv,  especially 
after  the  fluid  has  been  evaporated.  A  similarly  colored  mirror  is 
obtained,  which  gradually  fades  on  cooling. 

The  delicacy  of  the  test  is  the  same  as  that  of  Giinzburg. 

Examination  for  the  Presence  of  Combined  Hydrochloric  Acid. — 
The  presence  of  combined  hydrochloric  acid  cannot  be  demonstrated 
by  means  of  simple  tests  like  those  just  described,  but  is  inferred 
indirectly,  as  shown  in  the  following  method  : 

Separate  Estimation  of  the  Free  and  Combined  Hydrochloric  Acid 
of  the  Gastric  Contents. — Toffee's  Method. — The  total  acidity  of 
a  given  amount  of  the  gastric  contents  is  first  determined,  as 
already  described,  and  termed  A.  This  indicates  the  amount  of 
the  physiologically  active  hydrochloric  acid,  viz.,  the  free  and  the 
com])ined  hydrochloric  acid,  as  well  as  that  of  any  acid  salts  and 
organic  acids  that  may  be  present. 

In  a  second  .specimen  the  total  amount  of  free  acids  and  acid 
salts  is  determined  by  titrating,  as  before,  with  a  -^  normal  solu- 
tion of  sodium  hydrate,  but  using  a  few  drops  of  a  1  per  cent, 
aqueous  solution  of  alizarin  (alizarin-monosulphonate  of  .<t>dium)  as 
an  indicator.  The  titration  is  carried  to  a  point  where  a  pure  violet 
color  is  obtained.  The  result  is  termed  B.  The  diflerence  between 
A  and  B  is  thus  referable  to  the  presence  of  the  combined  hydro- 
chloric acid,  and  termed  C. 

In  a  third  s])ecimen  the  amount  of  free  hydrochloric  acid  is 
determined  by  titrating  with  the  decinormal  solution  of  sodium 
hydrate,  using  a  few  drops  of  a  0.5  per  cent,  alcoholic  solution  of 
dimethyl-amido-azobenzol  as  indicator,  until  the  red  color  >vhioh 
first  appears  has  changed  to  yellow.  The  result  is  termed  F. 
F  plus  C  will  then  represent  the  amount  of  physiologically  active 
hydrochloric  acid  P,  viz.,  the  combined  and  free  acid,  while  the 
difference  between  A  and  P  corresponds  to  the  acid  salts  and 
organic  acids  that  may  also  be  present. 

^Iethod  of  Morxer  and  S.joqvist. — By  this  method  the  amount 
of  physiologically  active  hydrochloric  acid  can  also  be  estimated.  It 
is  somewhat  more  complicated  and  time-consuming  than  the  one  just 
described,  but  more  accurate.  It  is  based  upon  the  fact  that  on 
evaporating  the  gastric  contents  to  dryness  in  the  presence  of  barium 
carbonate,  and  subsequently   incinerating  the    residue,  the  organic 


2'HE  GASTRIC  JUICE.  133 

acids  are  destroyed,  while  the  hydrochloric  acid  combines  with  the 
barium,  and  can  thus  be  estimated  as  barium  chloride. 

To  this  end,  10  c.c.  of  the  hltered  stomach-contents  are  treated 
with  a  pincli  of  chemically  pure  barium  carbonate  and  evaporated  to 
dryness.  Tiie  residue  is  ignited  at  a  moderate  temperature  until 
white,  and  the  remaining  ash  extracted  with  hot  water.  After 
filtering  the  solution  (about  50  c.c.)  it  is  treated  with  an  equal  volume 
of  alcohol  (94  per  cent.)  and  0.75  c.c.  of  a  10  per  cent,  solution  of 
sodium  acetate  in  dilute  acetic  acid  (10  per  cent,  solution).  Tho 
barium  is  then  estimated  by  titrating  with  a  standardized  solution  of 
potassium  bichromate,  containing  8.5  grammes  of  the  chemically 
pure  substance  in  the  liter,  and  using  tetramethyl-paraphenyldiamin 
paper  as  an  indicator,  until  a  drop  of  the  titrated  fluid  causes  a  distinct 
blue  coloration  of  the  paper  within  one  minute.  From  the  number 
of  cubic  centimeters  employed  to  bring  about  the  end-reaction  the 
corresponding  amount  of  hydrochloric  acid  can  then  be  calculated 
by  multiplying  with  0.00405,  if  the  bichromate  solution  was 
standardized  with  a   j-^,j  normal  solution  of  barium  chloride. 

Method  of  Leo. — This  method  is  based  upon  the  observation 
that  calcium  carbonate  combines  with  free  and  loosely  bound  hydro- 
chloric acid  at  ordinary  temperatures  to  form  neutral  calcium  chlo- 
ride, while  the  acid  phosphates  are  not  affected.  If  then  the  total 
acidity  of  the  stomach-contents  is  first  determined,  and  tlie  acidity 
referable  to  acid  salts  deducted  from  this  figure,  the  amount  of 
physiologically  active  hydrochloric  acid  is  ascertained.  Organic 
acids,  of  course,  must  first  be  removed  by  extracting  with  ether 
(50-100  c.c.  for  10  c.c.  of  gastric  juice).  As  the  monophosphates  of 
potassium  and  sodium,  however,  are  changed  to  monocalcium  phos- 
phate in  the  presence  of  calcium  chloride,  which  requires  double  the 
quantity  of  sodium  hydrate  solution  for  its  neutralization  than  the 
corresponding  amoimt  of  the  alkaline  phosphates,  it  is  either  neces- 
sary to  divide  the  number  of  cubic  centimeters  of  the  sodium 
hydrate  solution  which  is  used  in  the  second  titration  by  2,  or  to 
make  the  first  titration  under  the  same  conditions  as  the  first,  viz., 
after  adding  an  excess  of  calcium  cldoride  solution. 

To  this  end,  then,  we  proceed  as  follows:  15  c.c.  of  the  filtered 
gastric  contents  are  treated  with  a  pinch  of  dry  and  chemically 
pure  calcium  carbonate.  The  mixture  is  well  stirred  and  passed  at 
once  through  a  dry  filter.  Ten  c.c,  from  which  the  carbon  dioxide 
is  expelled  by  a  current  of  air,  are  then  treated  with  5  c.c.  of  a  con- 
centrated solution  of  calcium  chloride  and  titrated  as  usual.  The 
resulting  value  is  termed  P,  and  represents  the  acid  phosphates. 
The  total  acidity  is  then  determined  in  another  specimen,  after 
adding  the  same  amount  of  the  calcium  chloride  solution,  and  the 
result  termed  T.  T  minus  A  will  then  represent  the  amount  of  the 
physiologically  active  hydrochloric  acid. 

The  combined  hydrochloric  acid  may,  of  course,  be  readily  deter- 
mined   with    either    of  the    two    methods  which    have   just    been 


134  THE  DIGESTIVE  FLUIDS. 

described,  by  sej^arately  estimating  the  amount  of  free  hych'ochloric 
acid  by  Topfer's  metliod,  and  deducting  the  result  from  the  total 
amount  of  the  physiologically  active  acid.  More  accurate  results 
are  probably  reached  in  this  manner  tlian  Avith  T(»])ier's  method, 
unless  some  experience  has  been  gained  in  the  titration  with  alizarin. 

Sliould  organic  acids  also  be  present,  their  amount  mav  be  esti- 
mated by  deducting  from  the  total  acidity  the  result  reached  with 
the  above  method. 

If  monophosphates  are  present  at  the  same  time,  the  resulting 
figures  will  be  a  little  too  low ;  but  the  error  Avhich  is  thus  incurred 
is  trifling.  It  may  be  obviated,  liowever,  Ijy  making  use  of  Leo's 
method  (see  abovej. 

Lactic  Acid. — Tests  for  Lactic  Acid. — In  order  to  assure  our- 
selves that  any  lactic  acid  that  may  be  found  in  the  gastric  contents 
has  not  been  introduced  into  the  stomach  from  without,  it  is  neces- 
sary to  make  such  examinations  after  the  administration  of  a  test- 
meal,  in  which  the  acid  in  question  does  not  occur  preformed.  The 
meal  which  is  almost  exclusively  used  for  this  purpose  in  clinical 
work  is  the  so-called  test-meal  of  Boas.  It  consists  of  a  plateful 
of  oatmeal  soup,  which  is  prepared  by  adding  a  tablespoonful  of 
rolled  oats  and  a  little  salt  to  a  liter  of  water,  and  boiling  down  to 
about  500  c.c.  The  contents  of  the  stomach  are  then  drawn  off 
after  one  hour,  filtered,  and  treated  as  described  l)elow. 

Uffelmaxn's  Test. — About  10  c.c.  of  the  filtered  crastric  con- 
tents  are  extracted  with  ether  (50-100  c.c.)  by  shaking  in  a  sepa- 
rating funnel  for  from  twenty  to  thirty  minutes.  The  ethereal  ex- 
tract is  then  evaporated  to  dryness  by  distilling  on  a  water-bath  ; 
the  residue  is  taken  up  with  a  few  cubic  centimeters  of  distilled 
water,  and  treated  as  follows  :  3  drops  of  a  saturated  aqueous  solu- 
tion of  ferric  chloride  are  mixed  with  an  ecpial  number  of  drops  of  a 
concentrated  solution  of  pure  carbolic  acid,  and  diluted  with  water 
until  a  light-amethyst  color  is  obtained.  To  this  solution  a  portion 
of  the  ethereal  extract  is  added,  when  in  tlie  presence  of  lactic  acid  a 
lemon  or  canary  color  develops. 

The  delicacy  of  the  test  is  such  that  the  presence  of  0.1  })er  cent, 
of  lactic  acid  can  be  demonstrated. 

Kelling's  Test. — Five  or  10  c.c.  of  the  filtered  stomach-contents 
are  diluted  from  ten  to  twenty  times  with  water  and  treated  with  1 
or  2  drops  of  a  5  per  cent,  aqueous  solution  of  ferric  chloride.  In 
the  presence  of  lactic  acid  a  distinct  greenisli-ycllow  color  is  obtained 
when  the  tu])e  is  held  to  the  light. 

Boas'  Test. — This  test  is  more  accurate  than  the  two  just  de- 
scribed, but  more  time-consuming  and  complicated.  It  is  based 
upon  the  decomposition  of  lactic  acid  into  formic  acid  and  acetic 
aldehyde,  and  the  demonstration  of  the  presence  of  the  latter.  To 
this  end,  from  10  to  20  c.c.  of  the  filtered  stomach-contents  are 
treated  with  a  slight  excess  of  barium  carbonate,  and  evaporated  on 
a   water-l^ath.     The  resulting  syrup  is  acidified  with  a  few  drops 


THE  GASTRIC  JUICE.  135 

of  phosphoric  acid,  and  freed  from  carljon  dioxide  by  momentary 
ebullition.  On  cooling-,  it  is  extracted  with  100  c.c.  of  ether  by 
shaking  for  about  thirty  minutes.  The  ethereal  extract  is  poured 
off,  the  etiier  distilled,  and  the  residue  taken  up  with  45  c.c.  of 
water.  After  filtering,  the  solution  is  then  treated  with  5  c.c.  of 
concentrated  sulphuric  acid  and  a  pinch  of  manganese  dioxide,  and 
carefully  heated  to  boiling.  Should  lactic  acid  be  present,  this  is 
now  decomposed,  and  acetic  aldehyde  liberated,  which  can  be  demon- 
strated bv  passing  the  vapor  into  a  test-tube  containing  Nessler's 
reagent  or  an  alkaline  solution  of  iodopotassic  iodide.  In  the  first 
instance,  yellowish-red  aldehyde  of  mercury  is  formed,  while  iodoform 
results  in  the  latter,  and  can  be  readily  recognized  from  its  odor, 
which  becomes  marked  when  the  solution  is  heated. 

Tests  for  Acetic  Acid  and  Butyric  Acid. — These  acids  can 
usually  be  recognized  by  their  odor.  Chemically  they  can  be 
demonstrated  as  follows  : 

Test  for  Acetic  Acid. — Ten  c.c.  of  the  filtered  stomach-contents 
are  extracted  with  ether  as  above.  The  ether  is  distilled  off,  the 
residue  taken  up  with  a  few  drops  of  water  and  accurately  neutral- 
ized with  sodium  hydrate.  To  this  solution  a  drop  or  two  of  a  very 
dilute  solution  of  ferric  chloride  is  added,  when  in  the  presence  of 
acetic  acid  a  dark-red  color  develops.  With  nitrate  of  silver,  on  the 
other  hand,  a  precipitate  is  obtained  which  is  soluble  in  hot  water. 

Test  for  Butyric  Acid. — The  ethereal  extract  of  10  c.c.  of  the 
stomach-contents  is  freed  from  ether  by  distillation,  the  residue  is 
dissolved  in  a  few  cubic  centimeters  of  water,  and  treated  with  a  trace 
of  calcium  chloride  in  substance.  In  the  presence  of  butyric  acid 
small  oil  droplets  separate  out,  the  nature  of  which  is  readily  recog- 
nized from  the  pungent  odor.  If,  in  the  place  of  calcium  chloride, 
a  slight  excess  of  baryta-water  is  used,  highly  refractive  rhombic 
platelets  or  granular,  wart-like  masses  are  obtained  on  evaporation, 
which  consist  of  barium  butyrate. 

Butyric  acid  can  also  be  recognized  by  the  peculiar  odf)r  of  pine- 
apple which  develops  when  the  dry  residue  of  the  ethereal  solution 
is  treated  with  a  little  sulphuric  acid  and  alcohol.  The  reaction  is 
due  to  the  formation  of  butyl  ethylate,  QH^Oo.CoHg. 

Quantitative  Estimation  of  Lactic  Acid. — This  is  best  accom- 
plished by  means  of  Boas^  method :  The  decomposition  of  the  lactic 
acid  is  effected  as  described  above.  After  the  addition  of  the  sul- 
phuric acid  and  manganese  dioxide  the  flask  is  closed  with  a  doubly 
perforated  stopper.  Through  one  aperture  a  bent  tube  passes  to  the 
condenser,  while  a  straight  tube  passes  through  the  other  opening, 
and  is  provided  at  its  free  end  with  a  small  piece  of  rubber  tubing 
that  is  clamped  ;  this  tube  should  dip  well  into  the  liquid,  and  serves 
for  passing  a  current  of  air  through  the  solution  when  the  distilla- 
tion is  completed.  The  mixture  is  then  distilled  until  about  four- 
fifths  of  the  contents  have  passed  over,  excessive  heat  being  carefully 
avoided,  so  as  to  prevent  decomposition  of  the  aldehyde.     The  dis- 


136  THE  DIGESTIVE  FLUIDS. 

tilliite,  which  is  received  in  a  high  Erlenmeyer  flask,  is  heated  with 
20  c.c.  of  a  y\|-  normal  solution  of  iodine  and  the  same  amount  of  a 
5.6  per  cent,  solution  of  potassium  hydrate.  The  mixture  is  thor- 
oughly shaken  and  set  aside  for  a  few  minutes.  The  excess  of 
iodine  is  then  estimated  after  adding  20  c.c.  of  hydrochloric  acid 
(specific  gravity  1.018),  by  titrating  with  a  Jq  normal  solution  of 
sodium  thiosulphate.  The  titration  is  carried  almost  to  the  point  of 
decolorization,  when  a  little  starch  solution  is  added,  and  the  titra- 
tion continued  until  the  last  trace  of  blue  has  disappeared.  Tlie  dif- 
ference between  the  number  of  cubic  centimeters  of  the  thiosulphate 
solution  employed  to  bring  about  this  end  and  the  amount  of  the  iodine 
solution  added,  viz.,  20,  will  indicate  the  number  of  cubic  centimeters 
of  the  latter  which  were  utilized  in  the  formation  of  iodoform.  By 
multiplying  this  number  by  0.003388  the  corresponding  amount 
of  lactic  acid  is  ascertained. 

Quantitative  Estimation  of  the  Organic  Acids. — The  organic  acids 
in  toto  may  be  estimated  by  one  of  the  methods  already  described 
(pages  132  and  133),  or  according  to  the  following  procedure,  as 
suggested  by  Hehner-Seemann.  This  method  is  based  upon  the 
transformation  of  the  oi'ganic  acids  into  their  alkaline  salts,  and 
their  subsequent  comlnistion,  with  the  formation  of  the  correspond- 
ing carbonates,  which  are  then  estimated  by  titration.  At  the  same 
time  the  amount  of  physiologically  active  hydrochloric  acid  can  be 
obtained  as  follows  :  10  c.c.  of  the  filtered  gastric  contents  are  neu- 
tralized with  a  decinormal  solution  of  sodium  hydrate,  and  the  total 
aciditv  thus  ascertained.  The  neutralized  solution  is  then  evaporated 
to  drvness  and  the  residue  incinerated.  Care  should  be  had,  however, 
that  the  application  of  heat  is  discontinued  as  soon  as  the  ash  has 
ceased  to  burn  with  a  luminous  flame.  The  residue  is  then  taken  up 
with  a  small  amount  of  water  and  titrated  with  a  decinormal  solution 
of  hydrochloric  acid.  The  number  of  cubic  centimeters  employed  to 
bring  about  the  end-reaction,  multiplied  by  0.00365,  will  indicate 
in  terms  of  hydrochloric  acid  the  amount  of  organic  acids  which  were 
originally  present.  By  deducting  this  value  from  the  total  acidity, 
as  first  ascertained,  the  amount  of  physiologically  active  hydro- 
chloric acid  is  found. 

The  Ferments  of  the  Gastric  Juice  and  their  Proenzymes. 

In  the  gastric  juice  of  almost  all  vertebrate  animals  two  fer- 
ments are  constantly  found.  These  are  termed  pepsin  and  chy- 
mosin,  or  rennin,  and  are  supposedly  furnished  by  the  so-called 
adelomorphous  or  central  cells  of  the  gastric  glands.  This  has  been 
established  by  resecting  the  pyloric  end  of  the  stomach  and  convert- 
ing it  into  a  blind  ]K)uch,  with  a  fistulous  o])ening  on  the  anterior 
abdominal  walls,  while  the  fundus  was  united  to  the  duodenum.  It 
was  then  noted  that  this  resected  portion  of  the  stomach,  in  which 
no  delomorphous  cells  are  fi)und,  furnished  an  alkaline  and  markedly 


THE  GASTRIC  JUICE.  137 

viscid  secretion,  which  contained  pepsin  and  large  amounts  of  mucin, 
but  no  hydrochloric  acid.  A  reversal  of  the  experiment,  on  the 
other  hand,  in  which  the  fundus  was  thus  isolated,  siiowed  that 
here  both  pepsin  and  hydrochloric  acid  are  secreted.  As  this 
portion  of  the  stomach  contains  both  delomorphous  and  adelo- 
morphous cells,  the  conclusion  naturally  suggested  itself  that  the 
hydrochloric  acid  is  furnished  by  the  delomorphous  cells,  while  the 
pepsin — and  the  same  apparently  holds  good  for  chymosin — is 
secreted  by  the  adelomorphous  cells.  The  latter  are  hence  also 
spoken  of  as  pepsin  cells,  while  the  delomorphous  cells  are  similarly 
termed  the  oxyntic  cells  of  the  stomach. 

While  this  view  has  been  generally  held  as  just  outlined,  it  has 
been  suggested  of  late  that,  after  all,  the  pyloric  end  of  the  stomach 
does  not  secrete  true  pepsin,  but  a  pseudopepsin,  which  is  capable 
of  acting  in  feebly  alkaline  as  well  as  in  acid  media,  and  yields 
tryptophan  among  the  products  of  digestion  to  which  it  gives  rise. 
Whether  or  not  the  pseudopepsin  also  occurs  in  the  cardiac  end  of 
the  stomach  has  not  been  definitely  ascertained,  but  seems  probable 
in  view  of  the  fact  that  an  aqueous  extract  of  that  portion  of  the 
stcMiiach  will  also  give  rise  to  the  formation  of  tryptophan  on  long- 
continued  digestion.  Our  knowledge  of  this  pseudopepsin,  how- 
ever, is  as  yet  not  very  extensive. 

In  addition  to  the  two  ferments  mentioned,  the  fat-splitting 
ferment  lipase  has  also  been  found  in  the  gastric  juice  of  man  and 
several  mammals.  (Volhard,  Kastle,  and  Loevenhart.)  It  is  appar- 
ently furnished  by  both  the  pyloric  portion  and  the  fundus,  but 
more  abundantly  so  in  the  latter  situation. 

As  in  the  case  of  the  ptyalin  of  the  saliva,  it  appears  that  the 
ferments  in  question  do  not  exist  in  the  cells  as  such,  but  in  the 
form  of  pro-enzymes  or  zymogens.  These  zymogens  in  the  case  of 
pepsin  and  chymosin  are  termed  propepsin  or  pepsinogen  and 
prorennin  or  chyniosinogen,  respectively. 

It  has  been  shown  that  an  extract  of  the  gastric  mucosa  of  a 
fasting  animal  when  treated  with  1  per  cent,  of  soda  and  main- 
tained aseptic  with  toluol  may  be  kept  for  months  without  losing 
its  digestive  capacity  ;  such  an  extract  may  subsequently  be  rendered 
physiologically  active  by  acidifying  with  hydrochloric  acid  to  the 
extent  of  0.2-0.3  per  cent. 

If  then,  however,  such  artificial  gastric  juice  is  neutralized,  and 
then  alkalinized  with  soda  to  the  extent  of  0.5  per  cent.,  the  solu- 
tion is  rendered  entirely  inactive  after  a  few  seconds,  when  warmed 
to  the  temperature  of  the  body,  and  it  is  to  be  noted  that  the  sub- 
sequent addition  of  hydrochloiic  acid  is  now  no  longer  capable  of 
restoring  the  activity  of  the  enzyme.  This  demonstrates,  of  course, 
that  while  the  proenzyme  is  more  or  less  resistent  to  soda,  the  fer- 
ment is  thereby  rapidly  destroyed.  On  the  other  hand,  it  apj)ears 
that  pepsin  is  more  resistent  to  the  influence  of  carbonic  acid  than  pro- 
pepsin.    Between  chymosin  and  its  zymogen  similar  relations  exist. 


138  THE  DIGESTIVE  FLUIDS. 

Of  the  chemical  nature  of  the  proenzymes  and  the  manner  in 
which  they  are  produced  by  the  cells,  practically  nothing  is  known. 
Xerve-influences,  no  doubt,  are  here  at  work,  as  in  the  case  of  the 
salivary  glands.  At  the  same  time  the  blood-supply  is  of  moment, 
and  we  find  that  during  the  process  of  digestion  the  blood-vessels  are 
dilated,  and  that  the  venous  circulation  is  more  rapid  and  the  blood 
of  a  light-red  color.  But  as  in  the  salivary  glands,  it  is  certain  that 
the  height  of  the  blood-pressure  has  only  indirectly  to  do  with  the 
activity  of  the  glands.  The  proenzymes  liere,  as  there,  are  formed 
through  a  specific  activity  on  the  part  of  the  cells,  from  food- 
material  which  is  supplied  by  the  lymph. 

A  solution  of  the  pro-enzymes  of  the  gastric  mucosa  that  has 
been  freed  from  albumins  does  not  give  the  common  reactions  of 
the  true  albumins.  From  such  solutions  the  pro-enzymes  are  not 
precipitated  by  dialysis,  but  it  is  interesting  to  note  that  on  long- 
continued  dialysis  they  are  rendered  inactive  ;  they  are  then  apjiar- 
ently  destroyed.  To  a  slight  extent  they  will  pass  througli 
Chamberland  filters,  as  well  as  those  of  Kitasato  ;  the  propepsin 
passes  through  somewhat  more  readily  than  tlie  prochymosin. 

Of  interest  also  is  the  tendency  of  the  proferments  to  adhere  to 
solid  substances,  and  it  has  been  shown  tliat  they  possess  selective 
properties  in  this  respect,  which  are  not  the  same  in  both  always. 
Lycopodium,  for  exam])le,  will  carry  down  the  pepsinogen,  but 
not  tlie  chymosinogen.  Charcoal,  powdered  marble,  and  calcium 
sub^iiate  will  carry  down  both. 

Whether  or  not  the  transformation  of  the  proenzymes  into  the 
corresponding  ferments  occurs  in  the  bodies  of  the  cells  has  not  been 
definitely  decided.  It  appears,  however,  that  in  the  majority  of 
animals  which  have  been  examined  in  tliis  direction  the  glands 
secrete  only  the  proenzymes,  and  that  these  are  then  rendered 
physiologically  active  by  the  hydrochloric  acid  of  the  gastric  juice. 
A  solution  of  propepsin,  which  may  be  obtained  by  macerating  in 
glycerin  the  mucous  membrane  of  a  fasting  animal,  is  thus  in  itself 
inert,  but  is  rendered  active  at  once  when  hyth'ochloric  acid  is  added 
to  the  extent  of  from  0.1  to  0.2  per  cent.  It  is  indeed  supposed 
that  pepsin,  which  in  itself  is  inactive  like  its  zymogen,  coiiibines 
w^ith  hydrochloric  acid,  which  alone  is  similarly  inert,  as  regards  its 
digestive  ability,  to  form  a  compound  acid,  the  so-called  jjepsin- 
hydrochloric  acid.  On  coming  in  contact  with  albuminous  mate- 
rial this  is  supposedly  decomposed,  with  the  formation  of  nascent 
hydrochloric  acid,  which  then  acts  as  the  active  digestive  principle, 
while  the  liberated  pepsin  combines  with  a  new  portion  of  hydro- 
chloric acid,  and  thus  serves  as  an  acid-carrier.  On  this  qnestion, 
however,  a  uniformity  of  opinion  does  not  exist ;  still,  the  hypoth- 
esis is  an  attractive  one,  and  has  a  good  deal  in  its  favor.  If  we 
thus  regard  the  action  of  a  ferment  as  essentially  influencing  the 
rapidity  of  reaction,  the  action   of   the    weak  hydrochloric  acid  of 


THE  GASTRIC  JUICE.   ■  139 

the  gastric  juice  could  be  compared  to  the  effect  of  stronger  solu- 
tions upon  albumins  under  the  application  of  iieat. 

Pepsin. — Pure  pepsin  occurs  in  the  form  of  minute  globules,  which 
resemble  the  globulites  of  egg-albumin,  but  are  somewhat  smaller. 
Their  diameter  does  not  exceed  15-20  jx.  They  are  not  doublv 
refracting.  The  substance  is  white  when  perfectly  pure  and  is 
not  hygroscopic.  It  is  soluble  in  water,  dilute  acids,  and  glycerin. 
From  its  aqueous  solutions  it  can  be  precipitated  by  half-saturatijn 
with  ammonium  sulphate,  and  it  is  also  thrown  down  on  dialysis. 

When  acidified  with  hydrochloric  acid  to  the  extent  of  from  0.1 
to  0.3  per  cent.,  it  is  capable  of  dissolving  albumins,  with  the  forma- 
tion of  albumoses  and  so-called  peptones  (see  below).  This  can 
readily  be  demonstrated  as  follows  :  an  artificial  gastric  juice  is 
prepared  by  dissolving  a  pinch  of  one  of  the  commercial  prepara- 
tions of  pepsin  in  dilute  liydrochloric  acid  (0.1-0.2  per  cent.)  to 
which  a  Hake  of  boiled  beef-fibrin  is  then  added,  The  mixture  is 
kept  at  a  temperature  of  about  40°  C,  when  it  will  be  noted  that 
after  a  short  time  the  fibrin  begins  to  swell  and  is  subsquently  dis- 
solved. In  the  solution  which  thus  results  albumoses  and  peptones 
can  be  demonstrated  (see  i)age  62).  Other  acids,  such  as  sulphuric 
acid,  nitric  acid,  phosphoric  acid,,  lactic  acid,  and  even  acetic  acid, 
are  also  capable  of  rendering  pepsin  physiologically  active,  but 
much  larger  amounts  of  these  are  necessary  to  bring  about  the 
same  result.  In  the  case  of  phosphoric  acid,  for  example,  an  acidity 
of  10—12  pro  mille  is  necessary.  Carbonic  acid  and  hydrocyanic 
acid,  on  the  other  hand,  are  without  effect.  Unlike  chymosin, 
ordinary  pepsin  does  not  bring  about  the  coagulation  of  casein,  but 
Pekelharing  has  shown  that  in  acid  solution  the  pure  substance  also 
coagulates  milk.  It  further  causes  the  precipitation  of  so-called 
plastein  (see  below)  in  concentrated  solutions  of  albumoses. 

In  neutral  and  alkaline  solutions  pepsin  is  inactive,  and,  as  has 
been  seen,  it  is  rapidly  destroyed  by  sodium  carbonate,  even  in  very 
small  amount.  Its  resistance  to  higher  temperatures  is  to  a  great 
extent  dependent  upon  the  reaction  of  its  solutions.  In  neutral 
solution  it  is  destroyed  at  55°  C.  ;  in  the  presence  of  0.2  per  cent, 
of  hydrochloric  acid  this  result  is  reached  only  at  65°  C,  and  in  the 
presence  of  jieptones  and  certain  salts  a  temperature  of  70°  C  is 
necessary  to  bring  about  the  same  end.  In  the  dry  state,  on  the 
other  hand,  the  ferment  may  be  heated  to  100°  C,  and  even  higher, 
without  being  destroyed.  At  temperatures  lower  than  40°  C.  pepsin' 
is  still  active,  but  less  energetically  so,  and  at  0°  C.  its  action  ceases 
altogether. 

Alcohol  precipitates  pepsin  from  its  solutions  without  affecting  its 
subsequent  activity,  unless  the  exposure  has  been  prolonged.  Some 
of  the  salts  of  the  heavy  metals,  such  as  the  acetates  of  lead  and 
platinum  (chloride,  as  also  tannic  acid,  magnesium  carbonate,  and 
ammonium  sulphate,  likewise  cause  the  precipitation  of  impure 
forms,   at  least,    but  are   without    effect    upon    the    ferment  itself. 


140  THE  DIGESTIVE  FLUIDS. 

Uranvl  acetate  is  an  excellent  reag^ent  for  the  preci]iitation  of" 
ferments  (and  albumins),  even  with  a  neutral  or  feebly  acid  reaction. 
Like  the  albumins,  pepsin  does  not  diffuse  through  animal  mem- 
branes, but  is  precipitated. 

To  a  certain  extent  the  rapidity  of  digestion  is  dependent  \\\Mm 
the  amount  of  pepsin  that  is  available ;  but,  as  in  the  case  of  all 
ferments,  very  small  quantities  are  sufficient  to  effect  an  amount  of 
chemical  change  that  is  apparently  out  of  all  proportion  to  the 
amount  present.  Thus,  Petit  claims  that  a  pepsin  preparation, 
which  he  prepared  himself,  was  capable  of  dissolving  500,000  times 
its  weight  of  fibrin  in  seven  hours.  The  much  more  impure 
commoricial  forms  are,  of  course,  far  less  active,  but  many  of  them 
possess  remarkable  digestive  power. 

The  ability  on  the  part  of  pepsin  to  digest  albumins  is,  however, 
limited ;  and  with  an  increase  in  the  amount  of  digestive  products 
formed,  its 'activity  gradually  diminishes  and  finally  ceases.  This 
can  be  obviated  in  a  measure  by  removing  these  products  as  they 
are  formed,  and  may  be  artificially  accomplished  by  allowing  the 
digestion  to  take  place  in  a  parchment  tube  which  has  been  sus- 
pended in  dilute  hydrochloric  acid.  The  peptones  which  are  formed 
then  pass  from  the  tube  by  dialysis,  and  in  this  manner  digestion 
can  be  carried  much  further  than  under  other  conditions.  Com- 
plete digestion,  however,  may  even  then  not  be  achieved. 

llegarding  tlie  chemical  nature  of  pepsin,  our  knowledge  has 
been  greatly  extended  through  the  researches  of  Pekelharing,  and 
Nencki  and  Sieber.  From  the  fact  that  it  is  possible  to  prej)are 
pepsin  solutions  which  actively  digest  albumin,  but  do  not  show 
the  common  albumin  reactions,  it  has  l)een  concluded  that  pure 
pepsin  is  probably  not  an  albumin.  The  researclies  of  the  investi- 
gators just  mentioned,  however,  seem  to  show  conclusively  that  it 
is  an  albumin  nevertheless,  although  it  cannot  be  classed  with  any 
of  the  known  forms.  It  contains  no  phosphorus,  but  nevertheless 
yields  xanthin-bases  on  hydrolysis  with  alkalies.  Both  Friedentluil 
and  Pekelliaring  have  shown  that  a  pentose  can  be  obtained  from 
it,  and  (piite  recently  the  latter  has  isolated  a  peculiar  acid  on 
hydrolysis  with  alkali  which  gives  the  biuret  and  xanthoproteic 
reaction,  as  also  that  of  Adamkiewicz  and  jNIillon.  This  acid  is 
termed  jjcpsiniG  acid,  and  is  derived  from  a  coagulation-product  of 
})epsin,  which  in  turn  is  formed  when  acid  solutions  of  the  pepsin 
are  rapidly  heated  over  tiie  free  flame.  J3oth  the  coagulation- 
product  as  also  the  pcpsinic  acid,  like  all  albumins,  are  Ijevorotatory. 
Elementary  analysis  of  the  pure  pepsin,  obtained  from  dogs  that 
had  been  operated  on  by  Pawlow's  method  ((x^sojihageal  and  gastric 
fistulse),  as  also  of  the  coagulation-product  and  the  acid,  gave  the 
followinj;  avera";e  results  : 

C 

Pepsin 51.i)<) 

Coaiijulatioii-pnxluc't.    .    .    .     50.3-') 
Pepsinic  acid 50.79 


H 

N 

S 

CI 

7.07 

14  44 

1.63 

0.49 

().<)8 

14.90 

1.G4 

7.02 

14.44 

1.08 

THE  GASTRIC  JUICE.  141 

From  the  mode  of  origin  of  pepsinic  acid  it  is,  of  course, 
clear  that  the  substance  can  contain  no  loosely  combined  suljihur. 
Noteworthy  is  the  fact  that  the  ]>epsin  contains  chlorine,  and  the 
evidence  appears  to  be  conclusive  that  the  chlorine  is  an  actual 
constituent  of  the  pepsin  molecule. 

Formalin  when  added  to  a  solution  of  pure  pepsin  to  the  extent 
of  2-3  ])er  cent,  produces  no  appreciable  effect  upon  the  digestive 
power  even  after  days.  While  pepsin  is  probably  constantly  found 
in  the  gastric  juice  of  adult  vertebrate  animals,  it  is  noteworthy 
that  it  is  absent  from  the  gastric  juice  of  sucking  pups,  as  shown  by 
Gmelin  ;  that  its  presence,  moreover,  is  not  essential  to  the  main- 
tenance of  life  is  shown  by  the  history  of  Czerny's  dog  and  Hoff- 
mann's observations  in  the  case  of  a  woman  whose  stomach  had  been 
removed  in  toto. 

Specific  tests  for  the  demonstration  of  the  pepsin  of  the  gastric 
juice,  as  compared  with  other  proteolytic  ferments  which  similarlv 
act  in  acid  solutions,  are  unknown.  As  a  result,  all  such  ferments 
have  been  designated  as  pepsin,  although  it  is  very  likely  that  they 
are  not  identical.  Such  ferments  have  been  observed  in  the  secre- 
tion of  the  glands  of  Brunner,  in  the  muscles,  the  kidneys,  the 
brain,  the  saliva,  and  the  urine. 

Isolation  of  Pepsin. — If  it  is  merely  desired  to  obtain  an  effective 
solution  of  pepsin  without  regard  to  the  purity  of  the  substance, 
the  following  procedure  may  be  employed  : 

v.  Wittich's  Method. — The  mucous  membrane  of  a  pig's 
stomach  is  carefully  dissected  off,  freed  from  mucus  by  washing 
with  water,  hashed,  rubbed  together  with  pure  (piartz  sand,  and 
finally  treated  with  glycerin,  containing  0.1  per  cent,  of 'hydrochloric 
acid,  in  the  proportion  of  10-20  grammes  for  1  part  of  mucous 
membrane.  The  mixture  is  kept  at  a  temperature  of  40°  C.  for 
from  one  to  two  weeks,  when  it  is  filtered.  The  extract  which  is 
thus  obtained  may  then  be  used  for  experimental  purposes  by 
diluting  with  0.1-0.4  per  cent,  of  hydrochloric  acid,  in  the  propor- 
tion of  2-3  :  100.  For  the  ])re))arati()n  of  a  ])ure  j)epsin  the  follow- 
ing method  should  be  emploved  : 

Pekelharixg's  Method. — Gastric  juice  uncontaminated  by 
food-material  or  saliva  is  obtained  from  dogs  that  have  been  operated 
according  to  Pawlow's  method  (stomach  and  oesophageal  fistulse). 
The  gastric  juice  obtained  in  portions  after  pseudofeeding,  is  filtered 
and  dialyzed  for  about  twenty  hours  against  distilled  water,  at  a 
temperature  not  much  above  0°  C.  A  portion  of  the  pepsin  is  thus 
]>recipitated  and  isolated  by  centrifugation.  It  is  collected  on  a 
filter,  washed  with  a  little  distilled  water,  and  dried  in  the  desiccator. 
The  liquid  ])ortion  of  the  original  material,  after  centrifugation  is 
half-saturated  with  ammonium  suljihate  (35  grammes  for  100  c c). 
The  resulting  precipitate  is  freed  from  salt  by  dialysis,  dissolved  in 
0.2  per  cent,  hydrochloric  acid,  reprecipitated  by  dialysis,  collected 
on  a  filter,  and  dried. 


142  THE  DIGESTIVE  FLUIDS. 

Quantitative  Estimation  of  Pepsin. — Accurate  methods  for  the 
quantitative  estimation  of  pepsin  are  not  available.  Relative  values, 
however,  can  be  obtained  by  the  following  method,  as  suggested  by 
Hammerschlag :  three  Esbach  tubes  (all)uminimeters)  are  em- 
ployed. Tube  1  is  filled  to  the  mark  U  with  a  mixture  of  10  c.c. 
of  a  1  per  cent,  solution  of  serum-albumin  in  0.4  per  cent,  of 
hydrochloric  acid  and  5  c.c.  of  filtered  gastric  juice.  Tube  2, 
which  is  the  standard,  is  filled  to  the  same  mark  with  the  serum- 
albumin  solution,  but  receives  in  addition  0.5  gramme  of  pepsin. 
Tube  3  contains  merely  10  c.c.  of  the  albumin  solution  and  5  c.c. 
of  distilled  water.  Esbach's  rear/ent,  which  consists  of  10  grammes 
of  picric  acitl  and  20  grammes  of  citric  acid,  dissolved  in  1000 
c.c.  of  distilled  water,  is  then  added  to  each  tube  to  the  mark  R. 
After  twenty-four  hours  the  amount  of  precipitate  is  read  oif 
and  the  difference  between  tubes  1  and  3  compared  with  that  of 
tube  2. 

Still  better  is  the  following  method,  reconmiended  by  Mett : 

Capillary  glass  tubes  are  prepared  measuring  from  1  to  2  mm.  in 
diameter.  They  are  filled  with  white  of  egg,  which  is  coagulated 
in  the  tubes  at  a  temperature  of  95°  C  The  tubes  are  then  cut  into 
pieces  from  1  to  2  cm.  long  and  placed  in  the  digestive  mixture  to 
be  examined.  Tiie  length  of  the  column  digested  in  a  given  length 
of  time  serves  as  a  measure  of  the  digestive  })ower  of  the  specimen 
examined.  In  practice  this  column  should  be  measured  in  milli- 
metres with  the  aid  of  a  magnify ing-glass.  The  calculation  of  the 
corresponding  amount  of  ferment  is  based  upon  the  law  of  Schiitz 
and  Borissow,  viz.,  that  the  corresponding  amounts  of  ferment  in 
two  solutions  bear  tiie  same  ratio  toward  each  other  as  tiie  square 
of  the  number  of  millimetres  of  the  column  of  egg-albumin  which 
has  been  dissolved  in  the  same  length  of  time. 

Example. — The  gastric  juice  of  a  normal  individual  is  procured 
at  the  height  of  digestion  after  giving  Ewald's  test  breakfast.  The 
tube  is  digested  for  thirty  minutes ;  at  the  end  of  this  time  the 
height  of  the  column  of  albumin  digested  measures  3  mm.  Then 
the  stomach  contents  of  a  second  individual  are  obtained  (the 
jiatient's)  and  similarly  treated  ;  in  this  case  the  column  of  digested 
albumin  measures  2  mm.  The  corresponding  amounts  of  ferment 
are  then  as  9  is  to  4. 

Pepsinogen. — The  presence  of  pepsinogen  in  tho  gastric  juice 
can  be  ascertained  only  when  hydrochloric  acid  is  abs(>nt,  as  it  is 
otherwise  transformed  into  tlie  active  enzyme.  Its  occurrence,  as 
such,  is  hence  essentially  a  pathologic  ]ihcnomenon,and  indicates  the 
absence  of  free  hydrochloric  acid.  But  while  the  latter  may  be 
absent  in  many  diseases  which  are  not  associated  with  structural 
abnormalities  of  the  gastric  mucous  membrane,  pepsinogen,  and  con- 
se(piently  also  pepsin,  are  found  lacking  only  in  disease  of  the 
stomach  itself,  and  when  complete  atrophy  of  the  glandular  struct- 
ures  has   occurred. 


THE  GASTRIC  JUICE.  143 

Test  for  Pepsinogen. — Specimens  of  gastric  juice  in  which  pepsin- 
ogen only  is  present  are  incapable  of  digesting  albumins.  In  such 
caseSj  as  I  have  just  said,  hydrochloric  acid  is  absent.  If  then 
the  solution  is  acidified  to  the  extent  of  from  0.1  to  0.3  per 
cent.,  and  the  dissolution  of  a  flake  of  boiled  beef-fibrin  now  occurs, 
the  presence  of  the  zymogen   may  be  inferred. 

Quantitative  Estimation  of  Pepsinogen. — The  determination  of  the 
absolute  quantity  of  pepsinogen  in  the  gastric  juice,  as  that  of  pep- 
sin, is  not  possible.  Relative  values,  however,  may  be  obtained  by 
the  following  method,  as  suggested  by  Boas  :  To  this  end,  specimens 
of  the  gastric  juice  are  variously  diluted  with  distilled  water  in  the 
proportion  of  1  :  5,  1  :  10,  1  :  20,  etc.  A  known  quantity  of  coagu- 
lated egg-albumin  or  serum-albumin  is  then  added  to  each  tube, 
as  also  1  or  2  drops  of  a  0.3  per  cent,  solution  of  hydrochloric  acid, 
for  every  10  c.c.  The  tubes  are  kept  at  a  temperature  of  about 
40°  C,  Avhen  the  degree  of  dilution  is  noted  at  Avhich  the  albumin  is 
still  dissolved.  The  greater  the  degree  of  dilution  at  which  this 
occurs,  of  course  the  greater  is  the  amount  of  pepsinogen — that  is, 
of  pepsin — present. 

Chymosin. — Chymosin,  or  rennin,  according  to  the  researches  of 
Glassner,  is  formed  only  in  the  glands  of  the  fundus,  and,  as  in 
the  case  of  pepsin,  is  secreted  in  the  form  of  a  pro-enzyme,  which, 
like  the  pepsinogen,  is  then  transformed  into  the  corresponding  fer- 
ment by  the  hydrochloric  acid  of  the  gastric  juice.  It  is  to  be 
noted,  however,  that  calcium  chloride  or  any  other  soluble  calcium 
salt  is  likewise  capable  of  bringing  about  this  result.  The  specific 
action  of  chymosin  is  exerted  upon  milk  or  lime-containing  solutions 
of  casein,  which  are  coagulated  in  neutral  and  even  feebly  alkaline 
solutions.  Unlike  pepsin,  chymosin  is  not  a  proteolytic  ferment, 
and  its  action  ceases  with  the  formation  of  paracasein.  It  is  there- 
fore surprising  to  note  that  chymosin  is  found  not  t)nly  in  the 
stomachs  of. mammals,  but  also  in  other  vertebrate  animals,  and 
even  in  certain  plants,  where  casein  as  a  food-stuif  certainly  does  not 
enter  into  consideration.  Our  knowledge  of  ferments  in  general,  how- 
ever, is  as  yet  very  defective,  and,  as  a  matter  of  fact,  we  are  acquainted 
only  with  the  more  manifest  reactions  of  these  bodies,  while  it  is 
quite  possible  that  they  possess  other  important  properties  of  Mhich 
we  are  now  in  ignorance.  It  is  conceivable,  moreover,  that  differ- 
ent varieties  of  chymosin  exist,  which,  as  a  class,  are  all  capable 
of  coagulating  casein,  but  which  differ  from  each  other  in  other 
respects  and  serve  other  purjioses. 

While  chymosin  is  also  active  in  feebly  acid  solution,  it  is  gradu- 
ally destroyed  at  a  temperature  of  from  37°  to  40°  C.  when  exposed 
to  the  action  of  gastric  juice  containing  0,3  per  cent,  of  hydrochloric 
acid.  The  ferment  is  here  apparently  digested  by  the  pepsin,  and  it 
is  thus  easily  possible  to  obtain  solutions  of  pepsin  which  are  alto- 
gether free  from  chvmosin.  In  neutral  solution  it  is  more  resistant, 
and  can  be  heated  to  a  temperature  of  50°  C. ;  at  70°  C,  however, 


144  THE  DIGESTIVE  FLUIDS. 

it  becomes  permanently  inactive.  In  its  dry  state,  on  the  other 
hand,  it  can  be  heated  to  110°  C.  without  losing  its  activity.  Alka- 
lies when  present  beyond  traces  destroy  the  substance,  as  tliev  do 
pepsin.  Like  all  other  ferments,  it  is  capable  of  effecting  an  exten- 
sive reaction,  even  when  present  in  small  amount.  The  quantitv  of 
ferment  contained  in  1  gramme  of  the  dried  and  pulverized  mucous 
membrane  of  the  fourth  stomach  of  the  calf,  when  dissolved  in 
water,  is  thus  capable  of  coagulating  200  liters  of  milk  in  one 
minute  at  a  temperature  of  50°  C. 

Of  the  chemical  nature  of  chymosin,  nothing  is  known ;  but,  as 
in  the  case  of  pepsin,  the  purer  preparations  do  not  give  the  usual 
reactions  of  albumins.  It  is  precipitated  from  its  neutral  solutions 
by  subacetate  of  lead,  as  also  by  urauyl  acetate,  wbile  the  acetate  of 
lead  and  tannic  acid  are  without  effect.  Alcohol  likewise  precijji- 
tates  the  ferment  and  gradually  renders  it  inactive.  Like  pepsin, 
it  is  not  dialyzable. 

Under  normal  conditions  chymosin  is  always  present  in  the  gastric 
juice  of  man.  In  certain  diseases  of  the  stomach,  however,  which 
are  associated  with  the  death  of  its  glandular  elements,  the  ferment,  as 
also  its  zymogen,  is  lacking. 

Tests  for  Chyinosm  and  Chymosinogen. — To  test  for  the  jiresence 
of  chymosin.  5  or  10  c.c.  of  milk  are  treated  with  a  few  drops  of 
the  filtered  gastric  juice  and  kept  at  a  temperature  of  from  37°  to 
40^^  C.  If  coagulation  occurs  within  ten  or  fifteen  minutes,  the 
presence  of  chymosin  may  be  assumed.  Should  the  gastric  juice, 
however,  be  markedly  acid,  it  is  necessary  first  to  neutralize  it  with 
barium  carbonate. 

To  test  for  chymosinogen,  the  milk  is  treated  with  2-3  c.c.  of  a 
1  per  cent,  solution  of  calcium  chloride,  and  10  c.c.  of  filtered 
gastric  juice  which  has  been  rendered  feebly  alkaline  with  sodium 
hydrate.  The  mixture  is  kept  at  a  temperature  of  from  37°  to  40° 
C,  when  in  the  presence  of  the  zymogen  a  thick  cake  of  casein  is 
fi)rmed  ^vithiu  a  few  minutes. 

Isolation  of  Chymosin. — To  isolate  chymosin  in  comparatively 
pure  form,  the  following  method,  as  sugire-ted  V)v  Hammarsten,  may 
be  employed  :  The  mucous  membrane  of  the  fourth  stomach  pf  the 
calf  is  carefully  dissected  off,  washed  with  water,  and  extracted  Avith 
an  0.1  per  cent,  solution  of  hydrochloric  acid,  as  already  described. 
The  infusion  is  then  neutralized  and  repeatedly  shaken  with 
]wwdered  magnesium  carbonate  until  the  pepsin  has  been  removed. 
The  filtrate  is  treated  with  subacetate  of  lead,  the  precipitate 
decomposed  with  verjf  dilute  sulphuric  acid,  and  the  acid  filtrate 
further  precipitated  with  an  aqueous  solution  of  stearin  soap.  The 
ferment  is  thus  thrown  down  together  with  the  fatty  acids,  from 
which  it  is  then  sejiarated  by  suspending  the  precipitate  in  water 
and  extracting  the  fatty  acids  with  ether.  The  chymosin  remains  in 
aqueous  solution,  and  may  now  l)e  precipitated  with  strong  alcohol. 
It  is  then  rapidly  collected  on  a  filter  and  dried. 


THE  GASTRIC  JUICE.  145 

Quantitative  Estimation  of  Chymosin  and  Chymosinogen. — As  in 
the  case  of  pepsin  and  pe])sinogen,  relative  values  only  can  be 
obtained.  The  gastric  juice  is  neutralized  with  a  very  dilute  solu- 
tion of  sodium  hydrate.  Tubes  are  then  prepared,  containing  5  or 
10  c.c.  of  the  gastric  juice,  variously  diluted  in  the  proportion  of 
1  :  10,  1  :  20,  1  :  30,  etc.,  to  which  an  equal  volume  of  neutral  or 
amphoteric  milk  is  further  added.  These  tubes  are  kept  at  a 
temperature  of  from  37°  to  40°  C,  when  the  degree  of  dilution  is 
noted  at  which  coagulation  still  occurs.  Under  normal  conditions 
a  positive  reaction  can  thus  be  obtained  in  man  with  a  degree  of 
dilution  varying  between  1  :  30  and  1  :  40. 

In  the  case  of  the  zymogen,  the  gastric  juice  is  rendered  feebly 
alkaline,  when  tubes  are  prepared  as  just  described.  Normally  a 
positive  reaction  can  thus  still  be  obtained  with  a  dilution  varying 
between   1  :  100  and  1  :  150. 

Other  Constituents  of  the  Gastric  Juice. — Of  other  con- 
stituents, the  gastric  juice  normally  contains  traces  of  sulpho- 
cyanides,  Mhicli  are  secreted  by  the  stomach  itself;  a  variable 
amount  of  mucin ;  a  small  amount  of  coagulable  albumin,  or,  if  the 
fluid  has  stood  for  some  time,  a  corresponding  quantity  of  albumoses 
or  peptones,  and,  as  already  shown,  certain  mineral  salts. 

Tlie  gases  which  are  found  in  the  stomach  have  in  part  been 
swallowed  with  the  food.  A  small  portion  is  further  referable  to 
eructations  from  the  duodenum,  while  a  third  portion  is  probably 
secreted  by  the  stomach  itself.  This  is  true  more  especially  of 
the  carbon  dioxide,  and  Schierbeck  has  shown  that  the  tension 
of  this  gas  gradually  increases  from  30  to  40  Hgmm.  while 
fasting,  to  130  to  140  Hgmm.  during  the  process  of  digestion, 
and  is  apparently  directly  proportionate  to  the  acidity  of  the 
gastric    juice. 

An  idea  of  the  relative  amounts  of  the  gases  which  are  normally 
found  in  the  stomach  may  be  formed  from  the  accompanying  table, 
which  is  taken  from  Planer : 

Man.  Dog. 

Vegetable  diet, 
vol.  per  cent. 

Carbon  dioxide 20.79-33.83 

Oxygen 0.37 

Nitrogen 72.50-33.22 

Hydrogen 6.71-27.58 

Other  gases,  such  as  marsh  gas,  olefiant  gas,  ammonia,  and  hy- 
drogen sulphide,  are  found  only  under  pathologic  conditions,  and 
are  referable  to  certain  fermentative  and  putrefactive  changes  which 
take  place  in  the  ingested  food. 

10 


Veg.  diet. 

Meat  diet. 

vol.  per  cent. 

vol.  per  cent. 

32.9 

25.2 

0.8 

6.1 

66.3 

68.7 

146  THE  DIGESTIVE  FLUIDS. 

THE  PANCREATIC  JUICE. 

As  has  been  pointed  out,  the  digestive  gUmds  which  have  so  far 
beeu  considered  are  not  essential  to  the  maintenance  of  life.  The 
salivary  glands  and  the  stomach,  moreover,  can  in  certain  animals 
be  eliminated  without  seriously  interfering  with  the  process  of 
digestion,  and  the  ferments  which  in  man  are  secreted  by  these 
structures  are  in  many  animals  absent.  The  pancreas,  on  the  other 
hand,  either  as  such  or  as  a  so-called  hepatopancreas,  is  found  in  all 
vertebrate  and  invertebrate  animals  in  wliich  the  process  of  diges- 
tion is  carried  on  in  a  well-defined  digestive  tube.  In  many, 
indeed,  it  represents  the  only  digestive  gland  of  the  body.  Its 
removal,  even  in  the  higher  animals,  invariably  leads  to  death.  In 
dogs,  in  which  this  operation  has  been  repeatedly  performed,  and  in 
which  life  may  go  on  for  a  few  weeks  thereafter,  it  has  beeu 
observed  that  as  a  result  of  such  interference  the  resorption  of  fats 
is  seriously  impeded,  so  that  practically  all  that  has  been  ingested 
reappears  in  the  feces.  In  the  case  of  the  albumins,  it  is  similarly 
found  that  but  44  per  cent,  is  absorbed,  and  of  the  ingested  starches 
from  20  to  40  per  cent,  is  eliminated  as  such.  Analogous  results 
are  obtained  in  the  human  being  where  atrophy  of  the  pancreas  is 
at  times  observed.  As  a  consequence,  rapid  emaciation  occurs,  and,  as 
has  been  stated,  death  ultimately  results.  It  appears,  however,  that 
the  fetal  issue  in  these  cases  is  not  exclusively  referable  to  impaired 
nutrition  as  a  result  of  defective  absorption.  It  is,  indeed,  possible 
to  counteract  this  effect  by  administering  a  sufficient  amount  of  raw 
pancreas  together  with  the  food,  whereby  the  resorption  of  both  fats 
and  albumins  is  greatly  i  inproved.  Death,  however,  takes  ])lace  never- 
theless. It  is  thus  apparent  that  besides  its  digestive  function  the 
pancreas  must  play  an  .additional  and  important  role  in  the  metab- 
olism of  the  animal  body.  We  find,  as  a  matter  of  fact,  that  fol- 
lowing the  extirpation  of  the  pancreas  in  dogs  a  severe  form  of  dia- 
betes rapidly  develops,  and  is  accompanied  by  the  appearance  of 
acetone,  diacetic  acid,  and  at  times  of  ,9-oxybutyric  acid  in  the  urine. 
That  this  is  not  due  to  suspension  of  the  pancreatic  digestion  can  be 
proved  in  various  ways.  If  the  animal  thus  receives  an  adequate 
amount  of  raw  pancreas  together  with  its  food,  the  absorption  of 
albumins  and  fits  is,  as  just  stated,  greatly  increased,  while  the 
diabetes  persists.  It  has  been  further  noted  that  ligation  of  the 
secretory  duct  does  not  lead  to  the  appearance  of  sugar  in  the  urine, 
and  that  the  diabetes  continues  after  extirpation  even  when  no  food 
is  consumed  for  several  days.  The  conclusion  hence  suggests  itself 
that  the  pancreas,  like  the  thyroid,  the  adrenal  body,  and  other 
glands,  prol)ablv  furnishes  an  internal  secretion  also,  which  in  some 
manner  controls  the  metabolism  of  glucose  within  the  animal  body. 
Arthaud  and  Butte,  it  is  true,  claim  that  diabetes  does  not  follow 
ligation  of  the  pancreatic  veins;  but  it  can  readily  be  imagined  tliat 
in  such  cases,  and  perhaps  even  under  normal  conditions,  the  internal 
secretion  of  the  gland  is  removed  through  the  lymph-channels.     It 


THE  PANCREATIC  JUICE.'  147 

has  been  shown,  moreover,  that  diabetes  does  not  occur  after  extir- 
pation of  the  i^ancreas  if  a  piece  of  the  gland  has  been  previously 
transplanted  under  the  skin. 

Of  the  nature  of  the  substance  which  is  thus  secreted  by  the 
pancreas,  and  in  the  presence  of  which  the  carbohydrate  metabolism 
continues  in  a  normal  manner,  our  knowledge  has  been  greatly 
extended  through  the  researches  of  Cohniieim  and  Hirsch,  who 
could  demonstrate  that  the  pancreas  furnishes  a  substance,  possibly 
of  the  nature  of  a  kinase,  wiiich  renders  possible  the  glucolysis  in 
both  muscle-tissue  and  the  liver.  In  its  absence  this  does  not  occur 
and  diabetes  is  the  necessary  consequence.  This  substance,  however, 
is  probably  not  furnished  by  the  pancreatic  cells  proper,  but  by  the 
cells  of  the  islands  of  Langerhans.  At  this  place  we  shall  deal 
only  with  the  pancreatic  secretion  proper. 

The  secretion  of  the  pancreatic  digestive  fluid,  like  that  of  the 
saliva,  is  ])artly  under  the  control  of  cerebrospinal  nerve-fibres,  which 
are  derived  from  the  vagus,  and  partly  of  sympathetic  fibres.  The 
material  from  which  the  secretion  is  elaborated  through  the  specific 
activity  of  the  glandular  cells  is  obtained  from  the  lymph,  and  ulti- 
mately, of  course,  from  the  blood.  In  carnivorous  animals,  in  which 
the  secretion  of  the  pancreas  is  intermittent  and  dependent  upon  the 
ingestion  of  food,  we  accordingly  find  that  in  its  stage  of  activity  the 
gland  assumes  a  bright  rose-color,  and  is  much  increased  in  size, 
while  in  the  resting  stage  it  is  pale  and  shrunken. 

General  Properties. — Fresh  pancreatic  juice  can  be  obtained 
only  by  establishing  an  artificial  fistula  in  the  pancreatic  duct.  But 
as  the  least  interference  with  the  integrity  of  the  gland  leads  at  once 
to  the  secretion  of  an  abnormal  fluid,  great  care  must  be  exercised 
to  operate  as  gently  and  rapidly  as  possible.  It  is  best  to  do  so 
about  three  hours  after  the  animal  has  received  a  large  meal.  The 
pancreatic  juice  which  is  then  obtained  represents  a  clear,  thick, 
colorless,  odorless,  and  very  concentrated  fluid  of  a  strongly  alkaline 
reaction,  which  does  not  show  any  proteolytic  activity,  however,  but 
can  be  rendered  physiologically  active  by  the  addition  of  intestinal 
juice  or  an  extract  of  the  intestinal  mucosa.  The  activating  factor 
is  a  peculiar  body  which  has  been  discovered  in  the  intestinal  juice 
by  Pawlow  and  his  pupils,  and  which  is  termed  enterokinase  (see 
page  156). 

Amount. — The  amount  of  pancreatic  juice  which  is  secreted  in 
the  twenty-four  hours  is  variable,  and  largely  dependent  upon  the 
quantity  and  quality  of  the  food  ingested.  From  permanent  fistulse 
45-100  grammes  are  supposedly  secreted  pro  kilogramme  of  the 
animal.  This  figure,  however,  is  no  doubt  too  high,  and  Bidder  and 
Schmidt  have  estimated  that  under  normal  conditions  the  secretion 
probably  does  not  exceed  5  grammes  pro  kilo. 

In  a  recent  case  of  pancreatic  fistula,  observed  in  man,  Pfaff  re- 
ports that  600  c.c.  of  the  secretion  were  obtained  in  twenty-four 
hours.  This  figure  thus  more  nearly  corresponds  to  the  results 
reached  by  Bidder  and  Schmidt. 


148  THE  DIGESTIVE  FLUIDS. 

Specific  Gravity. — The  specific  gravity  of  the  pancreatic  juice 
varies  between  1.008  and  1.010,  corres]x)nding  in  man  to  the  pres- 
ence of  from  2.5  to  7  per  cent,  of  solids.  "When  kept  for  a  few 
hours  at  ordinary  temperatures  it  loses  its  viscosity  and  transparency, 
and  rajiidlv  underfroes  putrefaction.  Crystals  are  then  deposited 
which  consist  of  lencin  and  tyrosin ;  they  result  from  the  digestion 
and  subsequent  decomposition  of  contained  albumins.  To  prevent 
these  changes,  the  secretion  must  be  placed  on  ice  at  once,  or  treated 
with  chloroform-water  or  a  similar  antiseptic  solution.  Owing  to 
the  presence  of  the  large  amounts  of  all)umin  which  the  pancreatic 
juice  contains,  the  liquid  coagulates  to  a  dense  mass  Avhen  heated  to 
a  temperature  of  74°  C.  On  cooling  to  0°  C,  or  when  dropped  into 
water,  a  clot  is  formed,  which  redissolves  on  warming  the  solution 
or  on  adding  an  excess  of  sodium  chloride. 

Chemical  Composition. — A  general  idea  of  the  chemical  composi- 
tion of  the  pancreatic  juice  of  the  dog  may  be  formed  from  the 
accompanying  analyses,  which  are  taken  from  C.  Schmidt  and 
Kriiger.  In  these  the  essential  points  of  difference  which  exist 
between  the  normal  fluid,  as  compared  with  the  secretion  obtained 
from  permanent  fistulae,  are  well  shown.  I  have  further  added  two 
analyses  of  human  pancreatic  juice  which  were  made  by  Herter  and 
Zawadsky.  In  Herter's  case  an  accumulation  of  the  secretion  had 
taken  place  in  the  duct,  owing  to  a  pressure-stenosis,  which  was  pro- 
duced by  a  carcinomatous  growth  ;  while  Zawadsky's  material  was 
obtained  from  a  pancreatic  fistula  which  had  remained  after  removal 
of  a  pancreatic  tumor. 

Of  the  two  secretions,  the  first  is  manifestly  abnormal  (see  below), 
while  the  analytical  results  in  the  second  case  conform  closely  with 
the  figures  which  were  ol)tained  by  Schmidt  in  what  may  be  regarded 
as  the  normal  pancreatic  juice  of  the  dog. 

I  have  finally  appended  an  analysis  of  the  contents  of  a  pancreatic 
cyst,  which  in  its  general   composition  resembles  the   secretion  ob 
tained  from  permanent  fistulse  in  animals.     Trypsin,  however,  was 
absent. 

Secretion  from  a  Xom-  Secretion  from  a  pcr- 

porary  fistula  of  the  manent  fistula  of  the 

dog.  dog. 

(C.  Schmidt.)  (Kriiger.) 

Water 900.8 980.44 

Solids  . 99.2 19.60 

Albumins,  peptones,  and  1  fio  *>  9  43 
ferments.                       T        '    ' 

Organic     matter,     comprising  1 

leucin,  tyrosin,   xanthins,  [•  30.4 3.3 

soaps,  and  fats.  J 

Mineral  constituents 8.8 3.o7 

Sodinm  chloride 7.35 0.93 

Potassium  chloride    ....  0.02 0.07 

Phosphate  of  calcium   ...  0.41 0.01 

Phosphate  of  magnesium  .    .  0.12 0.02 

Lime  and  magnesia  ....  0.32 2.53 


THE  PANCREATIC  JUICE.  149 


Human.  Human. 

(Herter's  case.)  (Zawadsky's  case.) 

Water 975.9 864.05 

Solids 24.2 135.95 

Peptones  and  enzymes  )                     -.-.  ^  no  nt; 

(no  albumin)  j-    .    .    .    •     il.o yZ.U£> 

Alcoholic  extract 6.4 43.90 

Mineral  ash 6.2 3.44 

Analysis  of  the  Contents  of  a  Pancreatic  Cyst  (Lenakcic), 

Water 928.1 

Solids 17.9 

Organic  matter 10.05 

Mineral  ash 7.85 

The  Ferments  and  their  Zymogens. 

Like  the  ferments  wliich  are  furnished  by  the  salivary  inlands 
and  the  central  cells  of  the  gastric  glantls,  the  enzymes  of  the  pan- 
creas occur  also  in  the  pancreatic  cells  in  the  form  of  zymogens, 
which  are  subsequently  transformed  into  the  active  ferments.  If 
a  fresh  pancreas  is  thus  extracted  with  glycerin,  it  will  be  noted 
that  the  residting  extract  has  no  proteolytic  properties  whatever, 
while  an  extract  obtained  after  the  gland  has  been  hashed  and 
exposed  to  the  air  for  some  time,  or  an  aqueous  extract  of  the  fresh 
gland,  digests  albumins  with  ease.  If  the  fresh  gland,  further,  is 
hashed  and  briefly  treated  with  a  1  per  cent,  solution  of  acetic  acid, 
and  then  extracted  with  glycerin,  an  active  preparation  is  obtained 
at  once.  Similar  results  are  reached  in  the  case  of  the  pancreatic 
ptyalin.  If  a  fresh  pancreas  is  thus  repeatedly  extracted  with 
glycerin  until  ptyalin  no  longer  passes  into  solution,  and  the  gland 
is  then  washed  in  water  and  left  exposed  to  the  air  for  a  short  time, 
an  additional  amount  of  the  ferment  can  be  obtained  l)y  further 
extraction  with  the  same  reagent.  In  what  manner  the  transforma- 
tion of  trypsinogen,  ptyalinogen,  steapsinogen,  and  the  other  zymo- 
gens, into  the  corresponding  ferments  is  normally  effected,  is 
unknown.  As  has  been  seen,  this  can  be  brought  about  artificially 
through  the  influence  of  watei',  dilute  acetic  acid,  and  probably  also 
through  the  activity  of  acid-forming  bacteria  or  the  oxygen  of  the  air. 

The  only  case  in  which  normal  })ancreatic  juice  has  been  obtained 
from  the  human  being  is  reported  by  Gliissner.  The  total  amount 
of  twenty-four  hours  varied  between  700  and  900  c.c. ;  the  largest 
secretion  took  place  during  digestion,  and  increased  steadily  until 
the  fourth  or  fifth  hour,  when  a  gradual  decrease  occurred.  The 
amount  secreted  while  fasting  represented  from  one-third  to  a  half 
of  the  total  amount.  The  quantity  was  very  materially  influenced 
by  the  ingestion  of  hydrochloric  acid,  increasing  to  twice  the  amount. 
The  secretion  was  absolutely  devoid  of  any  proteolytic  properties, 
but  manifestly  contained  the  proferment  of  trypsin,  as  it  could  be 
readily  activated  by  intestinal  juice.  Gliissner  ascertained  that  in 
the  fasting  condition  the  trypsinogen   secretion  is  practically  zero. 


150  THE  DIGESTIVE  FLUIDS. 

but  that  it  slowly  increases  from  the  time  when  food  is  ingested, 
to  reach  its  maximum  in  the  fourth  or  fifth  hour,  after  which  it 
again  decreases  to  the  eighth  hour.  Parallel  with  the  amount  of 
trypsinogen  runs  the  alkalinity  curve.  The  fresh  pancreatic  juice 
has  marked  lipolytic  properties,  which  are  increased  by  both  the 
bile  and  the  enteric  juice,  such  that  with  a  mixture  of  the  three 
secretions  the  activity  is  from  four  to  five  times  as  great  as  in  the 
case  of  the  pancreatic  juice  alone.  I^ike  the  proteolytic  power,  so 
also  does  the  lijiolytic  power  of  the  pancreatic  juice  reach  its  maxi- 
mum in  the  fourth  hour.  In  addition  the  pancreatic  juice  has 
marked  diastatic  properties,  but  it  is  incapable  of  further  decompos- 
ing the  resulting  maltose,  which  is  effected  by  the  enteric  juice. 
Neither  a  lactase  nor  an  invertin  is  found  in  the  human  pancreatic 
juice  (see  page  155). 

In  chronic  fistulse  conditions  are  different,  and  the  question  whether 
or  not  the  digestive  ferments  are  ])resent  depends  here  upon  the 
character  of  the  stimulus  given,  viz.,  upon  the  character  of  the  food. 
With  an  exclusive  meat  diet  trypsin  appears  as  such,  and  only  as 
such.  With  one  of  milk  and  bread  the  zymogen  only  appears,  and 
must  first  be  activated  by  the  enteric  juice.  The  amylolytic  fer- 
ment, on  the  other  hand,  is  not  influenced  in  this  manner,  and  is 
always  secreted  as  ferment.  With  steapsin  conditions  resemble 
those  in  the  case  of  trypsin.  With  a  diet  of  carbohydrates  and 
much  fat  the  zymogen  appears,  while  with  a  meat  diet  the  ferment 
is  furnished  directly  (Lintwarew).  The  activating  principle,  as  I 
have  stated,  is  a  peculiar  body  which  Pawlow  and  his  pupils  have 
demonstrated  in  the  intestinal  juice,  and  which  is  termed  entero- 
kinase  (see  page  156). 

Of  the  chemical  composition  of  the  zymogens  we  know  little,  but 
it  appears  that  they  are  of  an  albuminous  nature  and  of  a  more 
complex  composition  than  the  ferments  themselves.  On  decomposi- 
tion trypsinogen  thus  yields  the  corresponding  ferment  and  an 
unknown  albuminous  substance.  Of  the  origin  of  the  zymogens, 
and  of  the  manner  in  which  they  are  produced  in  the  cells,  we  know 
nothing. 

Trypsin. — Trypsin  is  the  most  important  proteolytic  ferment 
which  is  found  in  the  animal  world,  and  in  its  action  on  albumins  is 
fully  capable  of  replacing  pepsin  when  this  is  absent.  Its  digestive 
power,  moreover,  is  much  more  extensive  than  that  of  pej^sin,  and  it 
is  further  capable  of  decomposing  albumins  to  amido-acids  and 
hexon  bases.  Its  hydrolytic  effect  may  thus  be  compared  to  the 
action  of  strong  mineral  acids  under  the  application  of  heat.  This 
explains  also  the  fac-t  that  leucin  and  tyrosin  are  so  frequently  found 
in  the  pancreatic  juice.  The  extensive  digestive  activity  of  trypsin 
is  well  shown  when  the  gland  is  finely  hashed  and  treated  with  a 
large  amount  of  chloroform-water,  so  as  to  guard  against  putrefac- 
tive changes.  When  kept  at  a  temperature  of  40°  C.  autodigestion 
rapidly  takes  place,  and  after  several  days  it  will  be  noted  that  while 


THE  PANCREATIC  JUICE.  151 

trypsin  is  still  present  in  its  full  activity,  the  other  ferments  have 
disappeared.  Together  with  the  various  albumins  of  the  gland  they 
have  apparently  been  digested  by  the  more  powerful  ferment. 

While  trvpsin  acts  most  energetically  in  feebly  alkaline  or  neutral 
solutions  (0.25-1.0  per  cent,  of  sodium  carbonate),  it  is  also  capable 
of  digesting  albumins  in  slightly  acid  media,  providing  that  the 
acidity  is  not  due  to  the  presence  of  a  free  mineral  acid ;  the  diges- 
tive process  is  under  sucii  conditions,  however,  much  less  active. 
Free  mineral  acids  rapidly  destroy  the  ferment.  Its  optimum  tem- 
perature lies  between  37^  and  40°  C.  In  neutral  solution  it  is 
destroyed  at  45°  C,  while  in  feebly  alkaline  media,  and  especially 
in  the  presence  of  albumoses  and  certain  ammoniacal  salts,  it  can  be 
heated  somewhat  higher  without  impairment  of  its  digestive  power. 

One  of  the  most  important  characteristics  of  trypsin  is  its  inability 
to  transform  tirmly  combined  nitrogen  into  the  loosely  combined 
form.  On  long-continued  tryptic  digestion  of  serum-albumin  it 
will  thus  be  noted  that  the  amount  of  nitrogen  which  can  be 
obtained  on  distillation  with  magnesia  is  essentially  the  same  as  the 
amount  that  can  be  obtained  on  hydrolysis  with  acids. 

As  in  the  case  of  pepsin,  the  digestive  effect  of  trypsin  is  to  a 
certain  extent  dependent  upon  the  amount  of  the  ferment  present, 
and  here  as  there  the  action  of  the  enzyme  ultimately  ceases 
when  the  digestive  products  accumulate  beyond  a  certain  amount. 
Impure  extracts  can  be  prepared,  as  has  been  pointed  ont,  by 
extracting  the  gland  with  glycerin  after  it  has  been  left  exposed  to 
the  air.  The  purer  forms,  on  the  other  hand,  which  can  be  obtained 
according  to  Kiihne's  method  (see  below),  are  insoluble  in  glycerin 
and  alcohol,  but  soluble  in  water. 

Of  the  chemical  nature  of  trypsin  little  is  known.  In  acid  solu- 
tion it  is  coagulated  by  heat,  and,  according  to  Kiiline,  decomposed 
into  an  albumin  and  peptone.  An  analysis  of  a  fairly  pure  prepara- 
tion has  given  the  following  results  :  carbon,  52.75  percent.;  hydro- 
gen, 7.51;  nitrogen,  16.55;  oxygen  plus  sulphur,  23.19  (Loew). 
Its  composition  is  thus  very  similar  to  that  of  the  peptones,  and  it  is 
hence  possible  that,  unlike  the  other  digestive  ferments  which  we 
have  thus  far  considered,  trypsin  may  be  an  albuminous  substance. 

Test  for  Trypsin. — The  test  for  trypsin  resolves  itself  into  the 
demonstration  of  the  presence  of  a  proteolytic  ferment  which  is 
capable  of  digesting  albumins  in  alkaline  solution,  with  the  ultimate 
formation  of  amido-acids.  To  this  end,  the  solution  in  question  is 
rendered  alkaline  with  sodium  carbonate  to  the  extent  of  from  0.25 
to  1.0  per  cent.  A  small  flake  of  fibrin  is  then  added  and  an 
amount  of  thymol  sufficient  to  saturate  the  solution,  so  as  to  guard 
against  putrefactive  processes.  The  mixture  is  kept  at  a  tempera- 
ture of  40°  C.  If  the  fibrin  is  then  dissolved  and  leucin  or  tyrosin 
can  be  demonstrated  in  the  resulting  solution,  the  presence  of  trypsin 
may  bo  inferred.  To  this  end,  it  is  only  necessary  to  evaporate  the 
solution  to  a  thick  syrup  and   to  examine  this  microscopically  (see 


152  THE  DIGESTIVE  FLUIDS. 

page  207).  Should  the  solution  to  be  tested  contain  a  free  mineral 
acid,  or  larg;er  amounts  of  organic  acids,  no  result  "svill,  of  course,  be 
obtained,  as  the  trypsin  has  then  been  destroyed. 

Isolation  of  Trypsin. — Unless  it  is  desired  to  obtain  try])sin  in  as 
pure  a  form  as  possible,  alkaline  solutions  of  the  common  pancrcatin 
preparations  which  are  sold  in  the  shops  can  be  used  for  experimental 
purposes.  Otherwise  the  method  of  Iviihne,  as  modified  by  Gautier, 
should  be  employed  :  The  fresh  pancreas  is  finely  hashed  and 
washed  with  ice-cold  water,  to  which  1  pro  mille  of  salicylic  acid 
has  been  added.  After  four  hours  the  mass  is  treated  with  a 
large  amount  of  a  5  pro  mille  solution  of  sodium  carbonate, 
containing  an  excess  of  thymol,  and  is  kept  for  twelve  hours  at  a 
temperature  of  from  37°  to  40°  C.  The  acid  and  alkaline  extracts 
are  then  mixed,  treated  with  0.5  per  cent,  of  sodium  carbonate, 
filtered,  feebly  acidulated  Avith  acetic  acid,  and  saturated  with 
ammonium  sulphate  in  substance.  The  precipitate  which  then 
separates  out  contains  all  the  trypsin.  It  is  dissolved  in  water, 
filtered,  and  dialyzed,  so  as  to  remove  the  salt  which  is  still  present, 
as  also  traces  of  peptones  and  leucin.  The  resulting  solution  is 
concentrated  at  a  very  low  temperature,  and  the  trypsin  finally  pre- 
cipitated A\ith  strong  alcohol.  It  is  then  rapidly  filtered  off  and 
dried.  The  amount  of  material  which  can  thus  be  obtained  from 
one  gland  is  always  very  small,  but  sufficient  to  digest  a  large 
quantity  of  albumin  wdien  dissolved  in  about  100  c.c.  of  water. 

The  so-called  dry  pancreas  of  Kiihne,  from  which  trypsin  can 
likewise  be  obtained,  and  which  is  also  used  in  digestive  experiments 
as  such,  is  prepared  by  extracting  the  fresh  gland  with  alcohol  and 
subsequently  Avitli  ether  until  it  is  free  from  fats.  The  remaining 
material,  which  contains  the  active  ferment,  is  then  dried  and  pul- 
verized, and  can  be  kept  in  this  form  indefinitely. 

The  Amylolytic  Ferment  of  the  Pancreatic  Juice  (Amylop- 
sin). — The  amylolytic  ferment  of  the  pancreatic  juice  is  l)y  many 
thought  to  be  identical  with  the  ptyalin  of  the  saliva.  It  can  be 
isolated,  according  to  Gautier's  method,  from  an  aqueous  infusion  of 
the  fresh  gland  that  has  remained  exposed  to  the  air  for  about 
twenty-four  hours,  as  already  described.  To  demonstrate  its  action, 
a  few  cubic  centimeters  of  such  an  infusion,  or  a  glycerin  extract  of 
the  gland,  are  added  to  a  small  amount  of  starch  paste  and  kept  at  a 
temperature  of  40°  C.  The  mixture  is  then  tested  for  the  presence 
of  maltose,  as  already  described. 

Steapsin. — It  has  long  been  known  that  the  pancreatic  juice  pos- 
sesses the  powder  of  emulsifying  fats,  and  of  decomposing  these  into 
glycerin  and  the  corresponding  fatty  acids.  While  this  phenomenon 
has  by  s(jme  been  referred  to  the  action  of  bacteria,  others  hold  that 
it  is  dependent  upon  the  presence  of  a  specific  ferment,  which  has 
been  termed  steajxsin.  That  the  latter  view  is  probably  the  correct 
one,  ap])ears  from  the  fact  that  the  same  result  is  obtained  if  the 
perfectly  fresh    gland   is    used   and    care  is  taken    to    prevent    the 


THE  SECRETION  OF  THE  GLANDS  OF  B RUNNER.         153 

action  of  inicro-orgiinisiiis  by  adding  a  small  amount  of  hydro- 
cyanic acid  or  of  mercuric  chloride.  Of  the  nature  of  the  ferment 
nothing  is  known,  but  it  is  manifestly  very  unstable,  as  extracts 
prepared  from  glands  that  have  been  left  exposed  to  the  air  for 
twenty-four  hours  are  perfectly  inert  when  brought  in  contact  with 
neutral  fats.  Apparently  it  is  digested  during  this  time  by  the 
trypsin.  To  demonstrate  the  action  of  steapsin  on  fats,  a  small 
amount  of  perfectly  fresh  pancreas  is  finely  hashed  and  dehydrated 
with  90  per  cent,  alcohol.  It  is  then  dried  between  filter-paper  and 
placed  in  an  ethereal  solution  of  neutral  butyrin  (Butterfett).  On 
evaporation  of  the  ether  the  remaining  material  is  kept  between  two 
watch-crystals  at  a  temperature  of  from  37°  to  40°  C,  when  after 
awhile  a  distinct  odor  of  butyric  acid  becomes  manifest  (CI.  Bernard). 
Or,  a  small  amount  of  ventral  fat  is  treated  with  a  few  cubic  centim- 
eters of  a  feebly  alkaline  glycerin  extract  of  the  fresh  gland  (9 
parts  of  glycerin  and  1  part  of  a  1  per  cent,  solution  for  each  gramme 
of  the  gland),  to  which  a  few  drops  of  tincture  of  litmus  are  added. 
If  this  mixture  is  then  kept  at  a  temperature  of  37°  C,  it  will  be 
noted  that  the  alkalinity  gradually  decreases,  and  the  reaction  finally 
becomes  acid,  owing  to  the  liberation  of  free  fatty  acids  (Hammar- 
sten). 

The  emulsifying  action  of  the  pancreatic  juice  is  owing  to  the 
decomposition  of  the  neutral  fats  and  the  subsequent  saponification 
of  the  resulting  acids  by  the  alkaline  salts  which  are  at  the  same 
time  present. 

Maltase. — The  presence  of  maltase  in  the  pancreatic  juice  has 
been  inferred  from  the  fact  that  small  amounts  of  glucose  are 
invariably  formed  during  the  action  of  the  aqueous  or  glycerin 
extract  of  the  gland  upon  starch.  Glassner,  however,  notes  that  in 
his  case  of  pancreatic  fistula  an  inversion  of  maltose  to  glucose 
could  not  be  observed  (see  above). 

Chymosin. — Cliymosin  can  be  demonstrated  by  adding  a  small 
amount  of  the  pancreatic  juice  of  the  ox,  pig,  or  sheep  to  milk, 
when  coagulation  results,  as  in  the  case  of  the  chymosin  of  the 
gastric  juice.  The  ferment,  however,  is  not  present  in  all  mammals; 
in  dogs,  for  example,  it  is  absent. 

THE  SECRETION  OF  THE  GLANDS  OF  BRUNNER. 

In  some  animals,  such  as  the  rabbit,  a  secretion  analogous  to  that 
of  the  pancreas  is  furnished  also  by  the  glands  of  Brunner,  which 
are  found  in  the  upper  portion  of  the  duodenum.  But  in  other 
animals,  such  as  the  dog  and  the  pig,  the  function  of  these  glands 
is  comparable  to  that  of  the  pyloric  glands  of  the  stomach.  The 
reaction  of  the  secretion  in  the  dog  is  alkaline ;  the  specific  gravity 
]. 005-1. 020.  The  amount  secreted  does  not  materially  exceed  1 
c.c.  per  hour,  and  is  not  influenced  by  the  state  of  digestion  or  the 
character  of  the  food.     Ponamorew  found  that  in  the  dog  the  fluid 


154  THE  DIGESTIVE  FLUIDS. 

only  digested  albumin,  after  being  acidified.  This  observation 
agrees  with  tiie  results  obtained  by  Griitzner  and  others.  Gliissner, 
however,  states  that  both  in  the  dog  and  the  ])ig  the  proteolytic 
ferment  will  digest  albumins  not  only  in  the  presence  of  acids  (0.2— 
0.3  per  cent,  hydrochloric  acid),  but  also  with  a  neutral  and  feebly 
alkaline  reaction  (0.5  per  cent,  sodium  carbonate).  The  proteolytic 
action  of  the  ferment  extends  to  the  formation  of  tryptophan.  The 
ferment  itself  cannot  be  obtained  by  the  uranyl  acetate  method  ;  its 
characteristics  are  thus  the  same  as  those  of  the  pseudopepsin  of  the 
pyloric  glands. 

A  diastatic  ferment  does  not  occur  in  the  secretion  of  Brunner's 
glands. 

THE  ENTERIC  JUICE. 

The  enteric  juice  is  essentially  the  secretory  product  of  the  glands 
of  Lieberkiihn,  which  are  found  imbedded  in  the  mucous  membrane 
of  the  small  intestine,  and  to  some  extent  also  in  the  large  intestine. 
It  may  be  procured  by  resecting  a  loop  of  the  intestine,  measuring 
about  0.15—0.20  meter  in  length,  and  closing  the  proximal  end, 
while  the  distal  end  is  sutured  to  the  abdominal  wall.  The  mes- 
entery, however,  is  allowed  to  remain,  so  that  the  nerve-  and  blood- 
supply  of  the  portion  which  has  thus  been  isolated  is  interfered  with 
as  little  as  possible.  Instead  of  thus  forming  a  blind  pouch,  as  was 
first  done  by  Thiry,  both  ends  of  the  resected  loop  may  also  be 
sutured  to  the  anterior  abdominal  wall.  Such  fistulse  were  first 
established  by  Vella,  and  they  accordingly  bear  his  name.  The  free 
ends  of  the  divided  gut  are  in  either  case  then  united  with  each 
other  and  the  abdominal  wound  closed. 

In  animals  wdiich  have  thus  been  operated  it  is  noted  that  after 
five  hours  following  the  ingestion  of  food  a  copious  secretion  of 
fluid  takes  place  in  the  small  intestine,  which  continues  for  alwut 
six  hours.  During  this  period  of  activity  the  mucous  membrane 
presents  a  rose-red  color,  while  it  is  pale  Avhen  at  rest.  As  in  the 
case  of  the  pancreas,  the  secretion  is  intermittent.  It  can  be  mani- 
festly excited  in  a  reflex  manner,  as  a  moderate  secretion  may  be 
observed  within  an  hour  after  the  ingestion  of  food — that  is,  at  a 
time  ^vhen  but  little  chyme  has  passed  into  the  small  intestine. 
Later,  when  the  food  has  passed  the  stomach,  its  presence  alone  or 
that  of  the  digestive  products  ^vhich  have  been  formed  already, 
apparently  excites  the  increased  secretion  Avhich  is  then  observed. 
This  may  be  further  increased  artificially  by  mechanical  and  espe- 
cially by  electrical  stimulation,  and  it  is  indeed  possible  to  cause  the 
secretion  of  enteric  juice  in  this  manner  even  at  a  time  when  diges- 
tion is  not  going  on. 

In  the  upper  portion  of  the  duodenum  of  the  dog  the  secretion  is 
said  to  be  small  in  amount,  mucoid,  and  jelly-like,  ^yhile  further  on 
it  becomes  more  fluid. 

As  obtained,  the  enteric  juice  always  contains  a  not  inconsiderable 


THE  ENTERIC  JUICE.  155 

* 

amount  of  mucus,  which  is  derived  from  the  goblet-cells  that  are 
found  along  the  entire  length  of  the  intestinal  canal.  The  small 
amorphous  flakes  which  are  always  found  in  the  secretion  consist 
entirely  of  mucus.  The  juice  itself,  which  can  be  separated  from 
the  greater  portion  of  the  mucus  by  filtration,  is  in  the  lower  por- 
tion of  the  small  intestine  a  thin  light-yellowish  fluid,  of  a  strongly 
alkaline  reaction.  This  is  largely  due  to  the  presence  of  consider- 
able amounts  of  sodium  carbonate,  and  we  accordingly  find  that  on 
the  addition  of  an  acid  effervescence  of  carbon  dioxide  occurs.  Its 
specific  gravity  in  the  dog  is  fairly  constant,  and  corresponds  to 
about  1.010-1.011. 

The  amount  of  solids  is  largely  dependent  upon  the  character  and 
the  quantity  of  the  food  ingested,  and  in  the  dog  may  vary  between 
12.2  and  24.1  pro  mille.  These  variations  are  mainly  referable  to 
the  presence  of  albumins,  which  are  always  found  in  the  enteric 
juice,  while  the  inorganic  constituents  are  fairly  constant.  A 
general  idea  of  the  cliemical  composition  of  the  secretion  may  be 
formed  from  the  accompanying  analyses  : 

Dog  Horse 

(Thiry).  (Colin). 

Water 97.59  per  cent.         98.10  per  cent. 

Solids 2.41  "  '•  1.90  "        " 

Albumins _  .    .  0.80  "  "    \         ^  j.   » 

Other  organic  matter  (mucin)  .  0.73  "  "     j 

Mineral  ash 0.88  "  "  1.4-3   "        " 

Sodium  carbonate    ....  0.40  "  " 

Sodium  chloride 0.48  "  " 

Enteric  juice  contains  small  amounts  of  an  albumin  which  is 
coagulated  only  with  difficulty  and  which  has  commonly  been 
regarded  as  a  mucin.  It  appears,  however,  that  the  substance  is  in 
reality  a  nucleo-albumin  (Kutscher),  and  that  pure  enteric  juice 
contains  no  mucin. 

Of  the  amount  of  enteric  juice  which  is  secreted  under  normal 
conditions  in  twenty-four  hours  we  know  but  little.  In  the  intestinal 
juice  of  a  })atient  with  an  intestinal  fistula  Hamburger  and  Hekma 
found  a  daily  secretion  of  50-125  c.c,  with  an  average  of  88  c.c. 
The  amount  was  the  largest  from  the  fourth  to  the  seventh  hour 
following  the  principal  meal  of  the  day.  Mechanical  stimulation 
more  than  doubled  the  flow.  In  disease,  and  notably  in  Asiatic 
cholera,  exceedingly  large  quantities  may  be  observed  ;  but  in  such 
cases  we  are  no  doubt  dealing  with  a  direct  transudation  from  tlie 
blood,  and  not  with  an  actual  secretory  product  of  the  cells.  On 
section  of  the  corresponding  nerves  hvperse(^retion  can  be  artificially 
brought  about.  This  may  be  compared  to  the  paralytic  saliva  which 
is  obtained  from  the  sublingual  gland  on  .section  of  the  chorda  and 
of  the  sympathetic  fibres  that  supply  the  gland. 

Of  ferments,  the  enteric  juice  contains  invertin,  ptyalin,  maltase, 
lipase,  and  in  some  animals  lactase.  In  the  human  adult  lactase  is 
apparently  obtained.     The  ptyalin  is  present  in   only  very  small 


156  THE  DIGESTIVE  FLUIDS. 

amounts,  while  maltase  is  quite  abundant.  The  presence  of  lactase 
seems  to  be  dependent  ujion  whether  or  not  tlie  animal  receives 
lactose  in  its  food.  Its  formation  thus  appears  to  occur  only  when 
there  is  actual  need  for  its  presence.  A  fat-splitting  ferment  and  a 
proteolytic  ferment  (analogous  to  pepsin  and  trypsin)  are  not  fur- 
nished by  the  glands  of  Lieberkiihn,  and  on  first  sight  it  would 
thus  appear  that  as  an  organ  of  digestion  the  small  intestine  is 
rather  unimportant.  This,  however,  is  only  apparently  the  case. 
As  a  matter  of  fact  the  epithelial  cells  furnish  two  substances  at 
least  which  are  of  fundamental  importance.  The  one  is  the  entero- 
kinase  to  which  I  have  already  referred  on  several  occasions,  which 
activates  the  trypsin  of  the  pancreatic  juice.  The  other  is  the  so- 
called  erepsin  of  Cohnheim,  which  is  characterized  by  the  fact  that 
it  is  incapable  of  splitting  albumins  to  albumoses,  but  causes  the 
further  cleavage  of  the  latter  to  amido-acids.  In  addition  the 
mucosa  of  the  small  intestine  seems  to  contain  a  substance  which, 
when  treated  with  hydrochloric  acid  and  injected  into  the  circula- 
tion, causes  a  secretion  of  pancreatic  juice.  The  inactive  principle 
is  termed  prosecretion  and  its  active  form  secretin. 

Enterokinase. — The  enterokinase  itself  has  no  digestive  power; 
it  merely  activates  the  trypsin  of  the  pancreatic  juice,  viz.,  it  trans- 
forms the  inactive  zymogen  into  the  active  enzyme.  Possibly  it 
exerts  a  similar  action  upon  ptyalin,  though  this  may  not  be  constant. 
Steapsin  is  not  influenced.  Very  interesting  is  the  fact  that  the 
secretion  of  the  kinase  can  only  be  effected  by  the  introduction  into 
the  intestinal  lumen  of  ixorinal  pancreatic  secretion.  Boiled  pancre- 
atic juice,  puj'ely  mechanical  stimulation,  as  also  the  injection  of 
pilocarpi n,  only  give  rise  to  the  secretion  of  an  enteric  juice,  which 
is  free  from  kinase.  The  kinase  is  destroyed  by  a  temperature  of 
67°  C.  Hamburger  and  Hekma,  who  were  able  to  demonstrate  the 
presence  of  enterokinase  in  the  enteric  juice  of  man,  also  ascertained 
that  the  substance  is  not  capable  of  activating  indefinite  quantities 
of  pancreatic  juice,  but  that  a  certain  quantity  of  enteric  juice  can 
only  activate  a  definite  amount  of  pancreatic  juice;  they  accordingly 
conclude  that  enterokinase  is  not  a  ferment,  and  they  propose  to 
term  the  substance  zymolysin. 

The  enterokinase  has  not  yet  been  obtained  as  such  ;  it  is  precipi- 
tated from  extracts  of  the  intestinal  mucosa  in  im]Hire  form  by 
means  of  acids.  It  closely  adheres  to  any  precipitated  nucleo- 
proteids. 

Delezenne  states  that  he  has  demonstrated  the  presence  of  entero- 
kinase also  in  the  leucocytes  of  the  blood,  and  he  explains  the  action 
of  zymogen-containing  pancreatic  juice  upon  fil)rin  without  an  extrn 
addition  of  enterokinase  on  the  basis  of  the  presence  of  leucocytes 
contained  in  the  meshes  of  the  fibrin  or  in  the  pancreatic  juice. 
The  same  writer  claims  to  have  found  enterokinase  in  bacteria,  in 
various  fungi  (Amanita),  and  in  snake-venom. 

Erepsin. — (This  is  considered  in  the  section  on  Resorption.) 


THE  BILE.  157 

Secretin  and  Prosecretin. — According  to  Bayliss  and  Starling, 
it  is  possil:)le  to  extract  a  substance  from  the  mucosa  of  the  duodenum 
and  jejunum  with  acids,  which  when  introduced  into  the  circukition 
causes  a  secretion  of  pancreatic  juice.  This  substance,  as  I  have 
already  indicated,  they  term  secretin,  and  it  is  supposedly  formed 
by  the  acids  from  an  inactive  product,  the  prosecretin,  which  exists 
as  such  in  the  cells.  The  secretin  is  not  destroved  bv  boiling:  in 
either  acid,  neutral,  or  alkaline  solutions,  and  accordingly  it  cannot 
be  a  ferment. 

Camus  has  further  ascertained  that  while  secretin  can  be  ob- 
tained by  various  organic  and  inorganic  acids  (not  with  boric  acid 
or  carbonic  acid),  a  more  active  preparation  is  obtained  with  hydro- 
chloric acid,  nitric  acid,  and  sulphuric  acid,  than  with  other  acids. 
When  once  formed,  it  retains  its  activity  even  after  being  neutralized 
or  alkalinized. 

Aside  from  its  action  upon  the  pancreatic  cells  secretin  is  also 
said  to  increase  the  secretion  of  bile  and  of  saliva.  Atropin  and 
anaesthetics,  notably  chloroform,  diminish  its  activity. 

Popielski  has  recently  found  that  secretin  can  also  be  obtained 
from  the  mucosa  of  the  rectum,  the  ileum,  the  stomach,  and  that  it 
can  even  be  isolated  from  the  arterial  blood.  It  appears,  moreover, 
from  his  researches  that  the  body  has  not  only  a  specific  action  so 
far  as  the  pancreatic  cells  are  concerned,  but  that  on  intravenous  or 
hypodermic  injection  it  stimulates  nearly  all  digestive  glands  to 
activity. 

THE  BILE. 

Formerly  it  was  generally  supposed  that  the  bile  played  an  im- 
portant part  in  the  process  of  digestion,  and  was  further  capable  of 
controlling  the  intensity  of  the  putrefactive  and  fermentative  proc- 
esses which  even  normally  take  place  in  the  lower  intestinal  tract. 
It  has  now  been  definitely  established,  however,  that,  aside  from  its 
emulsifying  action  upon  fats,  the  secretion  possesses  no  digestive 
properties  whatever,  and  is  likewise  without  elfect  upon  the  bacteria 
which  are  normally  found  in  the  intestinal  canal.  We  accordingly 
find  that  in  animals  and  in  man  the  processes  of  nutrition  are  in  no 
way  interfered  with  if  the  bile  is  prevented  from  entering  the 
digestive  tube,  but  is  carried  to  the  outside  through  the  establish- 
ment of  a  fistulous  opening  in  the  common  duct,  jn'ovided  that  food 
is  administered  which  contains  but  little  fat.  With  a  diet  consist- 
ing of  albumins  and  carbohydrates  digestion  thus  continues  unim- 
paired, and  the  animal  is  capable  of  maintaining  its  nitrogenous 
equilibrium  practically  as  before.  If  fats,  however,  are  given  at  the 
same  time  in  large  amounts,  more  or  less  serious  digestive  disturb- 
ances soon  develop,  and  the  animal  loses  weight.  In  such  cases  it 
has  been  ascertained  that  whereas  normally  from  2  to  10  per  cent, 
of  the  ingested  fat  is  eliminated  in  the  feces,  from  31  to  47  per  cent, 
now  escapes  resorption.     The   oifensive  gases  which  are  then  passed 


158  THE  DIGESTIVE  FLUIDS. 

by  the  animal  are  referable  to  an  increase  of  the  putrefactive  proc- 
esses in  the  intestines,  it  is  true.  This  is,  however,  not  owing  to 
the  absence  of  bile  per  se,  but  to  the  fact  that  the  unabsorbed  tats 
envelop  the  albuminous  material,  and  thus  prevent  its  further  diges- 
tion, so  that  in  the  lower  portion  of  the  digestive  canal,  where  the 
putrefjictive  processes  are  most  intense,  the  bacteria  find  an  increased 
amount  of  pabulum  at  their  disposal,  and  an  increase  of  the  putre- 
factive products  accordingly  results.  In  the  presence  of  bile,  on  the 
other  hand,  this  does  not  occur,  as  its  emulsifying  effect  upon  the 
fats  soon  leads  to  their  absorption,  and  thus  leaves  the  albumins 
exposed  to  the  action  of  the  digestive  juices,  and  to  their  resorption 
in  turn.  Indirectly,  it  can  thus  control  the  process  of  putrefaction, 
but  such  action  is  not  due  to  any  germicidal  or  antiseptic  properties 
of  its  own.  On  withdrawing  fats  from  the  food  and  giving  an  ade- 
quate supply  of  carbohydrates,  normal  relations  are  soon  re-estab- 
lished, though  the  bile  is  absent  as  before. 

The  bile  in  reality  represents  a  most  important  excretory  product 
of  the  animal  l)ody,  and  may  in  this  sense  be  compared  to  the 
urine.  It  appears,  moreover,  that  those  waste-products  which  are 
markedly  toxic  in  action,  and  could  not  be  carried  to  the  kidneys 
through  the  blood-current  without  seriously  disturbing  the  general 
health,  are  formed  in  the  liver  directly,  and  are  hence  removed 
through  separate  channels  in  the  l)ile. 

Substances  are  further  eliminated  in  this  manner  which,  like 
cholesterin,  are  insoluble  in  water,  and  could  hence  not  be  excreted 
by  the  kidneys. 

For  convenience'  sake,  however,  the  bile  is  described  at  this  place. 

Secretion. — As  found  in  the  gall-bladder,  the  bile  represents  the 
secretory  product  of  the  liver-cells  which  is  eliminated  into  the 
radicles  of  the  biliary  passages,  together  with  so-called  mucus  which 
is  derived  from  the  epithelial  lining  of  the  greater  trunks  and  the 
gall-bladder  itself.  Its  secretion  is  continuous,  but  liable  to  exacer- 
bations which  are  essentially  dependent  upon  the  ingestion  of  food. 
According  to  Heidenhain,  the  curve  of  secretion,  in  reference  to 
amount,  shows  two  periods  of  greatest  activity,  which  in  the  dog 
correspond  to  the  third  to  the  fifth  and  the  thirteenth  to  the  fifteenth 
hour,  respectively,  after  the  administration  of  food.  This  curve, 
however,  is  further  influenced  by  the  character  of  the  food  ingested. 
With  an  albuminous  diet  three  stages  of  maximal  secretion  are 
thus  noted  :  The  first  after  two  to  three  hours,  a  second  stage 
after  five  to  eight  hours,  and  a  third  stage  after  twelve  to  fourteen 
ht)urs.  With  a  diet  of  albumins  and  fats,  on  the  other  hand,  the 
period  of  greatest  secretion  occurs  after  eleven  to  twelve  hours, 
and  with  one  of  albumin  and  carbohydrates  after  from  nine  to 
fourteen  hours.  These  figures  have  reference  to  observations  which 
were  made  after  the  administration  of  only  one  meal  in  the  twenty- 
four  hours.  With  two  meals  analogous  results  are  obtained.  The 
individual  periods,   however,  are   shorter  }  and  as   the   number  of 


THE  BILE.  159 

feedings  is  increased  the  secretion  becomes  more  uniform,  so  that 
with  a  meal  every  two  hours  no  variations  of  moment  can  be 
discerned. 

Amount. — The  amount  of  bile  which  is  eliminated  in  the  twenty- 
four  hours  is  variable  even  under  normal  conditions.  In  dogs  from 
2.9  to  36.4  grammes  can  be  obtained  pro  kilogramme  of  weight  of 
the  animal.  In  man  the  secretion  apparently  varies  between  400 
and  800  grammes ;  but  it  is  possible  that  these  figures  do  not  repre- 
sent normal  values.  Ranke  has  estimated  that  a  man  weighing  75 
kilogrammes  secretes  about  1050  grammes  even  in  health.  To  a 
certain  extent  the  amount  is  influenced  by  the  character  of  the  food, 
and  it  appears  to  be  quite  generally  accepted  that  a  diet  rich  in 
albumins  will  call  forth  a  greater  secretion  of  bile,  but  it  is  note- 
worthy that  this  increased  secretion  does  not  occur  at  once,  but 
only  on  the  second  or  even  the  third  day.  The  carbohydrates  are 
thought  to  diminish  its  amount,  or  are  at  least  incapable  of  increas- 
ing this,  like  the  albumins.  The  fats  are  probably  without  effect 
in  either  direction. 

It  was  formerly  thought  that  a  number  of  drugs  could  increase 
the  flow  of  the  bile,  and  physicians  were  wont  to  administer 
cholagogues  when  they  supposed  that  the  secretion  of  bile  was 
deficient.  This  view  has  now  been  abandoned,  as  it  has  been  defi- 
nitely established  that  drugs  are  without  effect  in  this  direction. 
The  only  cholagogue,  indeed,  if  it  may  so  be  termed,  is  the  bile 
itself.  This  is  readily  understood,  if  we  bear  in  mind  that  the 
bile  is  essentially  an  excretory  product. 

General  Properties. — The  color  of  the  bile  differs  in  different 
animals,  and  may  vary  from  a  bright  yellow  to  an  intense  grass- 
green,  with  various  shades  of  brown  and  blue.  In  man  it  is  usually 
of  a  golden-yellow  color,  but  it  may  at  times  appear  bright  green 
even  when  perfectly  fresh. 

While  pure  bile,  uncontaminated  with  mucus,  is  a  thin  transparent 
fluid,  that  which  is  eliminated  into  the  intestinal  tract  and  is  found 
in  the  gall-bladder  is  markedly  viscid  and  more  or  less  turbid.  Its 
taste  is  intensely  bitter,  with  a  sweetish  and  nauseating  after-taste. 
The  odor  is  in  many  animals  peculiar,  and  at  times  suggestive  of 
musk.  The  reaction  is  normally  alkaline.  After  exposure  to  the 
air,  however,  it  soon  becomes  acid,  but  becomes  alkaline  again  owing 
to  the  development  of  trimethylamin  and  ammonia.  The  specific 
gravity  varies  between  1.010  and  1.033.  In  the  gall-bladder  it  is 
higher  (1.026-1.033),  where  a  constant  resorption  of  water  is  going 
on,  and  where  considerable  amounts  of  mucus  are  added  to  the  bile, 
than  in  the  bile  proper,  which  is  secreted  by  the  liver  (1.010-1.012). 
We  accordingly  find  that  the  amount  of  solids  is  greater  in  the 
bladder-bile  than  in  that  which  may  be   termed   the  hepatic  bile. 

Chemical  Composition. — The  following  analyses  show  the  gen- 
eral chemical  composition  of  the  bile  in  different  animals,  and  also 


IGO 


THE  DIGESTIVE  FLUIDS 


illustrate    the    differences    which    exist    between    bladder-bile   and 
hepatic  bile : 


Human  bladder-bile       Human    hepatic     bile    (normal), 
(normal).  (Frerichs.)  (Hammarsten,  1  to  3;  Zeynek,  4.) 


Water       

Solids 

^lucin       .... 
Pigments     .    .    . 
Salts  of  bile  acids 
Taurocholates 
Glycocholates 
Fatty  acids  (soaps) 
Cholesterin 
Lecithin       .    . 

Fat 

Soluble  salts 
Insoluble  salts 


1. 
860.0 
140.0 

26.6 

72.2 


1.6 

3.2 
6.5 


859.! 
140.! 

29.1 

91.' 


2.6 


.2} 
■'  { 


974.80 
25.20 

5.29 

9..31 
3.03 
6.27 
1.23 
0.63 

0.22 

8.07 
0.25 


964.74 
35.26 

4.29 

18.24 
2.17 

16.16 
1.36 
1.60 

/0.57 

(.  0.95 
6.76 
0.49 


3. 

974.60 

25.40 

5.15 

9.04 
2.18 
6.86 
1.01 
1.50 
0.65 
0.61 
7.25 
0.21 


Analyses  of  the  Bladder-bile  of  Animals. 


Dog. 
(Hoppe-Seyler.) 

"Water 813.56 


Solids 

Sodium  glycocholate 
Sodium  taurocholate 
Cholesterin       .... 

Fats       

Soaps    

Lecithin 

Mucin       

Other  oro^anic  solids;  \ 

insoluble  in  alcohol  ( 

Inorganic  solids  .    .    . 


186.44 
•        ) 

122.8  r 

2.911 
15.11  I 
16.03  I 
18.11  J 

3.49 

6.0 


Pig. 
(Grundelach- 
Slrecker.j 

888.0 
112.0 


83.8 


22. 


5.9 


Ox. 
(Berzelius.) 

904.4 
95.6 


80.0 


3.0 


12.6 


Birds. 
(Marsson.) 

800.2 
199.8 

170.6 


3.6 


25.6 


21.0 


4. 
989.24 
30.76 

2.08 

18.31 


2.08 

2.30' 

0.73 

9.10 
0.81 


Shad. 
(Schloss- 
berger.) 

944.8 
55.2 

36.3 


14.8 


Analyses  of  the  inorganic  .salts  have  pfiven  the  following  results 
which  are  taken  from  Jacobsen  and  Hoppe-Seyler,  respectively 
The  figures  have  reference  to  100  parts  by  Aveight  of  mineral  ash 


Sodium  chloride 

Potassium   chloride 

Sodium  carbonate 

Trisodium  phosphate 

Tricalcium  phosphate        .        

Calcium  carbonate 

Potassium  sui])hate 

Sodium  suli)hate 

Iron,  Silica      t 

Magnesium,  copper J 

'  Including  fat. 

-  This  figure  is  too  low,  owing  to  the  fact  that  Hoppe-Seyler's  analysis  has  reference  to  the 
inorganic  salts,  which  were  not  dissolved  by  alcohol. 


Man 

Ox 

(hepatic  bile). 

(bladder-bile). 

65.16 

7.50 ' 

3.39 

11.16 

2.50 

15.90 

4.44 

40.0 

9.50 

variable 

2.0 

25.0 

traces 


THE  BILE.  161 

The   Mucinous   Body   of  the   Biles. 

The  mucinous  body  which  is  found  in  the  bhidder-bile  of  all 
animals  is  apparently  of  a  different  nature  in  different  animals.  The 
mucus  of  the  human  bile  thus  largely  consists  of  true  mucin,  while 
in  ox-bile  a  mucinous  nucleo-albumin  is  principally  found.  To 
isolate  this  latter,  the  bile  is  precipitated  with  5  times  its  volume 
of  absolute  alcohol  and  immediately  centrifugalized.  The  super- 
natant liquid  is  poured  otf,  and  the  sediment  rapidly  dried  with 
filter-paper  and  dissolved  in  water.  By  repeating  this  process  the 
substance  can  be  obtained  in  a  fairly  pure  form.  Its  character  as 
a  nucleo-albumin  becomes  apparent  on  treating  its  neutral  solutions 
with  a  small  amount  of  hydrochloric  acid,  A  flocculent  precipitate 
then  develops,  which  readily  dissolves  upon  the  further  addition  of 
hydrochloric  acid  to  the  extent  of  0.3  per  cent.  This  solution  re- 
mains clear  for  a  long  time  even  when  kept  at  the  temperature  of 
the  body.  If  a  small  amount  of  pepsin,  however,  is  added,  a  sepa- 
ration of  nuclein  occurs.  On  fusing  the  dried  substance,  moreover, 
with  potassium  hydrate  and  sodium  nitrate,  an  amount  of  phosphoric 
acid  is  obtained  which  is  greatly  in  excess  of  the  amount  required 
to  saturate  all  of  the  mineral  ash  that  is  j^resent  when  calculated  as 
tricalcium  phosphate.  On  boiling  with  a  dilute  mineral  acid  no 
reducing-substance  is  formed,  as  in  the  case  of  true  mucin.  Acetic 
acid  precipitates  the  substance  from  its  solutions,  in  the  absence  of 
biliary  acids ;  but  this  precipitate,  unlike  that  of  mucin,  is  soluble 
in  an  excess  of  the  acid. 

The  mucin  proper  which  is  found  in  human  bile  may  be  isolated, 
as  already  described  (page  125). 

The   Biliary  Acids. 

The  biliary  acids  Avhich  are  normally  found  only  in  the  bile  are 
essentially  compound  amido-acids,  which  are  formed  through  the 
union  of  glycocoll  on  the  one  hand,  and  taurin  on  the  other,  with  a 
cholalic  acid.  In  the  bile  of  sharks  Hammarsten  discovered  the  ex- 
istence of  a  third  group  of  biliary  acids,  which  a'-e  rich  in  sulphur, 
and,  like  the  conjugate  sulphates  of  the  urine,  yield  sulphuric  acid 
on  boiling  with  hydrochloric  acid.  Traces  of  these  acids  are  said 
to  occur  also  in  human  bile.  Of  their  chemical  nature,  however, 
nothing  is  known. 

Under  pathologic  conditions,  when  the  free  outflow  of  bile  is 
impeded,  owing  to  a  swelling  of  the  mucous  membrane  of  the 
common  duct  or  to  the  presence  of  a  calculus,  resorption  of  the 
bile  takes  place  through  the  lymph-channels,  and  the  secretion 
thus  finds  its  way  into  the  blood.  Jaundice  then  results,  and  in 
such  cases  not  only  the  bile-pigments,  but  also  the  bile-acids  can 
be  demonstrated  in  the  general  circulation.  To  the  presence  of  the 
latter  is  due  the  slow  pulse  which  is  so  constantly  observed  in 
11 


162  THE  DIGESTIVE  FLUIDS. 

icterus.  At  the  same  time  the  bile-acids  bring  about  a  dissohition 
of  the  red  corpuscles,  and  thus  manifest  their  true  nature  as  excretory 
products. 

Regarding  the  origin  of  the  biliary  acids,  we  know  only  that 
they  are  formed  exclusively  in  the  liver,  but  of  the  manner  in 
which  their  formation  takes  ])lace  we  are  ignorant. 

The  amido-radicles  of  the  bile-acids  are  unquestionably  formed  in 
the  general  nitrogenous  metabolism  of  the  body,  and,  as  such,  are 
found  in  other  organs  besides  the  liver.  Glycocoll  is  then  to  a  cer- 
tain extent  further  oxidized,  and  contributes  to  the  furmation  of 
urea.  Of  the  ultimate  fate  of  taurin,  Ave  know  that  its  sulphur  can 
be  oxidized  to  sulphuric  acid  and  be  eliminated  in  the  urine  in  this 
form.  In  the  himian  being  traces  are  further  excreted  as  tauro- 
carbaminic  acid  ;  and  in  rabbits,  at  least,  its  hypodermic  injection 
leads  to  the  elimination  of  the  body  as  such.  It  is  thus  difficult  to 
understand  why  two  substances  like  these,  for  the  removal  of  w^hich 
the  animal  body  has  manifestly  other  means  at  its  disposal  than 
their  elimination  through  the  bile,  should  here  appear.  AVe  may 
imagine,  however,  that  the  formation  of  the  bile-acids  in  the  liver 
represents  a  conservation  of  energy  on  the  part  of  the  body,  and 
further  constitutes  a  reserve  mechanism  by  which  waste-products 
can  be  removed  in  a  state  of  incomplete  oxidation. 

Of  the  origin  of  the  non-nitrogenous  acids,  which  combine  with 
glycocoll  and  taurin  to  form  the  biliary  acids,  we  know  nothing. 
The  synthesis,  however,  manifestly  occurs  in  the  liver,  and  here 
only,  as  after  extirpation  of  the  organ  in  birds  and  frogs  bile-acids 
are  not  found  in  the  blood.  In  mammals,  also,  neither  bile- 
pigments  nor  bile-acids  can  here  be  demonstrated  after  ligating 
the  gall-duct  and  the  thoracic  duct. 

In  the  bile  of  most  animals  the  biliary  acids  occur  in  combination 
with  sodium,  while  in  sea-fish  they  are,  curiously  enough,  present  as 
potassium  salts. 

As  regards  the  relative  quantity  of  the  two  principal  biliary  acids 
which  are  fotuid  in  the  bile,  it  is  to  be  noted  that  in  man  glycocholic 
acid  is  usually  more  abundant  than  taurocholic  acid.  In  strictly 
carnivorous  animals,  on  the  other  hand,  the  latter  only  is  found  ; 
but  the  same  also  holds  good  for  certain  herbivora,  such  as  the  sheep 
and  the  goat.  In  other  animals  the  relation  is  variable,  and  in 
some,  it  is  stated,  glycocholic  acid  only  is  found. 

Isolation. — Collectively,  the  biliary  acids,  in  the  form  of  their 
salts,  can  be  obtained  in  the  following  manner :  the  bile  is  mixed 
with  animal  charcoal,  evaporated  to  dryness,  and  the  residue  ex- 
tracted with  absolute  alcohol.  This  extract,  which  contains  the 
biliary  acid  salts,  cholesterin,  fats,  soaps,  and  lecithin,  is  then 
filtered,  concentrated,  and  treated  with  a  large  excess  of  ether.  In 
this  manner  the  salts  of  the  bile-acids  are  precipitated,  while  the 
other  substances  remain  in  solution.  On  standing,  the  precipitate 
gradually  becomes  crystalline,  and  is  then  spoken  of  as  Platner's 


THE  BILE.  '  JG3 

erj/fif(ff!izfd  bile.  In  this  form  tlie  biliary  acids  are  conveniently- 
estimated  as  a  wliole.  If  then  it  is  desired  to  determine  the  rela- 
tive amount  of  the  two  jirincipal  acids  present,  it  is  only  necessary 
to  estimate  the  sulphur  in  Platner's  bile,  from  which  the  corre- 
sponding amount  of  taurocholic  acid  can  be  calculated. 

To  separate  the  two  acids  from  each  other,  Platner's  bile  is  dis- 
solved in  water  and  completely  precipitated  with  a  solution  of  neutral 
acetate  of  lead.  The  corresponding  lead  salts  are  thus  formed,  and 
can  now  be  readily  separated  from  each  other,  as  the  glycocholate  is 
thrown  down,  while  the  taurocholate  remains  in  solution.  In  the 
filtrate  the  latter  is  then  precipitated  with  ammonia.  To  obtain  the 
free  acid  from  the  glycocholate,  the  precipitated  lead  salt  is  sus- 
pended in  water  and  evaporated  to  dryness  in  the  presence  of  sodium 
carbonate.  The  sodium  salt  is  thus  obtained  and  extracted  with 
alcohol.  The  alcohol  is  evaporated  off,  the  residue  dissolved  in 
Avater  and  treated  with  hydrochloric  acid,  when  the  glycocholic  acid 
will  separate  out.  The  taurocholic  acid  can  be  similarly  obtained 
from  its  lead  salt  by  decomposing  the  solution  with  hydrogen  sul- 
phide. The  resulting  plumbic  sulphide  is  filtered  off,  the  filtrate 
evaporated  to  dryness,  the  residue  dissolved  with  a  small  amount 
of  alcohol,  and  then  precipitated  with  an  excess  of  ether,  when  the 
free  acid  is  thrown  down. 

Tests  for  the  Bile-acids. — Pettenkofer's  Test. — On  treating  the 
biliary  acids  in  aqueous  or  alcoholic  solution  with  a  few  drops  of  an 
0.1  per  cent,  solution  of  furfurol  and  1  or  2  c.c.  of  concentrated, 
chemically  pure  sulphuric  acid,  a  beautiful  purple  color  develops. 
As  furfurol  results  when  concentrated  sulphuric  acid  is  added  to 
a  carbohydrate,  the  test  may  also  be  conducted  by  treating  the  solu- 
tion of  the  biliary  acids  (1  or  2  c.c.)  with  a  few  drops  of  a  10  per 
cent,  solution  of  cane-sugar  and  then  Avith  sulphuric  acid.  In  either 
case,  however,  care  should  be  had  that  the  temperature  which  results 
during  the  reaction  does  not  exceed  70°  C,  as  otherwise  the  result- 
ing pigment  is  destroved.  This  may  be  obviated  by  substituting 
strong  phosphoric  acid  for  the  sulpluiric  acid,  and  placing  the  solu- 
tion in  boiling  water.  The  resulting  pigment  shows  two  bands  of 
absorption  on  spectroscopic  examination.  One  of  these  is  situated 
at  F ;  the  other  between  D  and  E,  near  E.  On  diluting  with 
alcohol  a  green  fluorescence  is  observed.  In  the  presence  of  an 
excess  the  red  pigment  disappears,  but  reappears  upon  the  further 
addition  of  sulphuric  acid.  On  standing,  the  color  gradually  turns 
to  a  bluish  violet. 

As  Pettenkofer's  reaction  can  also  be  obtained  with  other  sub- 
stances, such  as  the  phenols,  the  higher  alcohols,  certain  bases  of 
the  aromatic  series,  the  higher  hydrocarbons,  and  acids  of  the  fatty 
series  and  the  benzol  series,  it  is  always  necessary  to  isolate  previ- 
ously the  biliary  acids  as  Platner's  bile,  before  drawing  conclusions 
from  the  reaction.  Albumins,  if  present,  must  in  every  case  first 
be  removed.     For,  as  has  been  stated,  many  of  these  contain  carbo- 


164  THE  DIGESTIVE  FLUIDS. 

hydrate  gr()ii])s,  which  on  contaot  witli  ooneentrated  sul])]iun'o  arid 
give  rise  to  the  formation  of  furfuroL  This  in  turn  combines  Avith 
the  aromatic  oxy-acids  and  phenols  that  result  from  decomposition 
of  the  amido-acids  to  form  colored  compounds,  which  are  either 
identical  with  or  very  similar  to  those  obtained  in  the  case  of  the 
biliary  acids  (see  the  reaction  of  Adamkiewicz  and  the  hydrochloric 
acid  test  for  all^umins). 

Pettenkofer's  reaction  is,  in  the  case  of  the  biliary  acids,  referable 
to  the  non-nitrogenous  acid  constituents  of  these  acids,  viz.,  to 
cholalic  acid  or  one  of  its  congeners. 

Physiological  Test. — Aside  from  their  reaction  with  furfurol,  the 
bile-acids,  or  their  salts,  may  also  be  identified  as  such  from  their 
effect  upon  the  action  of  the  heart.  To  this  end,  the  heart  of  a  cura- 
rized  frog  is  exposed  and  moistened  with  a  small  drop  of  a  1  per 
cent,  solution  of  atropin,  so  as  to  eliminate  the  action  of  the  vagus. 
A  few  drops  of  an  aqueous  solution  of  the  bile-acids  are  then 
placed  upon  the  heart,  when  their  retarding  action  can  readily  be 
demonstrated. 

Glycocholic  Acid. — As  has  been  stated,  glycocholic  acid  is  the 
principal  biliary  acid  that  is  found  in  the  bile  of  man.  It  is  formed 
through  the  union  of  glycocoll  and  cholalic  acid,  as  represented  by 
the  equation  : 

Glycocoll.         Cholalic        Glycocholic 
acid.  acid. 

It  is  accordinglv  decomposed  into  these  constitutents  when  treated 
with  dilute  acids  or  alkalies  under  the  application  of  heat. 

In  the  pure  state  glycocholic  acid  occurs  in  the  form  of  fine,  silk- 
like needles,  which  are  readily  soluble  in  alcohol,  less  readily  so  in 
water,  and  insoluble  in  ether.  From  its  aqueous  solutions  it  is 
readily  precipitated  on  adding  a  small  amount  of  a  mineral  acid. 
The  crystals  melt  at  133°  C.  It  is  a  monobasic  acid,  and  forms 
salts  which,  with  the  exception  of  the  lead  and  silver  compounds, 
are  all  soluble  in  water  and  alcohol.  The  free  acid  and  its  salts 
give  Pettenkofer's  reaction,  and  turn  the  plane  of  polarization  to  the 
right. 

On  heating  glycocholic  acid  with  concentrated  sulphuric  acid  the 
anhydride  of  glycocholic  acid  is  precipitated  in  the  form  of  oily 
droplets,  which  subsequently  tend  to  coalesce.  This  anhydride  is 
termed  cholonic  acid,  and  has  the  composition  CagH^iXO^. 

Accordini;  to  Michailotf,  fflvcocholic  acid  when  treated  with  con- 
centrated  sulphuric  acid  in  the  presence  of  acetic  acid  is  said  to  yield 
an  orange  color  with  a  green  fluorescence.  On  salting  with  ammo- 
nium sulphate  a  precipitate  is  formed  which  in  its  reactions  is 
identical  with  biliverdin.  Urobilin  is  said  to  remain  in  solution. 
This  observation  is  of  special  interest,  as  showing  the  possible  rela- 
tionship which  may  exist  between  the  biliary  acids  and  the  bile- 
pigments.     ^Ye  find,  as    a    matter  of  fiict,  that  an  increase  in  the 


THE  BILE.  165 

production  of  bile-])igments  on  tlie  part  of  the  liver  is  associated 
with  a  diminished  formation  of  biliary  acids.  Others  have  con- 
cluded from  this  observation  that  a  connection  between  the  produc- 
tion of  bile-acids  and  bile-pigments  does  not  exist,  and  that  the 
origin  of  the  two  classes  of  substances  must  be  referred  to  a  separate 
activity  on  the  part  of  the  hepatic  cells.  It  appears  to  me,  however, 
that  this  inference  is  not  altogether  justifiable. 

Closely  related  to  the  common  glycocholic  acid  is  the  so-called  hyo- 
glycocholic  acid,  which  has  been  found  in  small  amounts  in  the  bile 
of  the  pig.  On  decomposition  it  yields  glycocoU  and  hyocholalic 
acid,  as  shown  in  the  equation  : 

C„H,3N05    +     HP    =    CoHjXOa    +    Cs^H^oO, 

Hyoglycocholic  GlycocoU.  Hyocholalic 

acid.  acid. 

The  substance  itself  is  almost  insoluble  in  M'ater,  but  soluble  in 
alcohol.  It  is  crystallizable,  but  usually  obtained  as  a  resinous 
mass.  Its  salts  are  precipitated  from  their  solutions  by  calcium 
chloride,  barium  chloride,  and  magnesium  chloride.  By  salting 
with  sodium  sulphate  they  separate  out  like  soaps.  Like  the  com- 
mon glycocholates,  they  give  Pettenkofer's  reaction. 

In  addition  to  these  two  forms  still  other  glycocholates  apparently 
exist.  In  the  bile  of  rodents  a  glycocholate  is  thus  found,  which 
cannot  be  salted  out  with  sodium  sulphate,  but  which  is  also  precipi- 
tated by  the  salts  of  the  alkaline  earths.  Of  its  nature,  however,  as 
also  of  the  so-called  gua)io-biliary  aczcly  which  apparently  belongs  to 
this  order,  nothing;  is  known. 

Isolation. — The  common  glycocholic  acid  is  most  conveniently 
obtained  by  starting  with  Platner's  bile,  that  has  been  prepared  from 
human  bile  or  from  that  of  the  ox,  as  already  described. 

Or,  the  following  method  may  be  employed  (Bleibtreu) :  Start- 
ing with  ox-bile  the  pigments  are  precipitated  with  uranium  acetate  ; 
the  bile  acids  remain  in  solution.  In  the  filtrate  glycocholic  acid  is 
precipitated  with  chloride  of  iron  ;  tlie  resulting  iron  compound  is 
decomposed  by  heating  with  ammonia,  the  hydroxide  of  iron  is  fil- 
tered otf,  the  filtrate  acidified  with  hydrochloric  acid  and  shaken 
with  ether ;  the  glycocholic  acid  separates  out  in  crystalline  form. 
Or,  to  purify  the  substance  further,  the  ammoniacal  solution  is  neu- 
tralized with  acetic  acid,  the  glycocholic  acid  precipitated  with 
uranium  nitrate,  and  the  precipitate  decomposed  by  iieating  with 
sodium  phosphate  .solution.  The  resulting  solution  of  the  sodium 
salt  is  acidified  with  hydrochloric  acid  and  shaken  with  ether,  when 
the  glycocholic  acid  separates  out. 

Hyoglycocholic  acid  can  be  isolated  from  the  bile  of  the  pig  by 
first  decolorizing  with  animal  charcoal  and  then  salting  with  sodium 
sulphate  in  substance.  The  acid  is  thus  precipitated,  and  can  then 
be  filtered  off.  It  is  washed  with  a  solution  of  the  salt,  dissolved  in 
water,  and  precipitated  in  the  form  of  the  free  acid  by  means  of 
hydrochloric  acid. 


166  THE  DIGESTIVE  FLUIDS. 

Taurocholic  Acid. — TaurochoHc  acid,  as  has  been  stated,  is  the 
only  biliary  acid  that  is  found  in  the  bile  of  the  purely  carnivor- 
ous animals,  but  it  also  occurs  in  the  bile  of  man  and  most  herbiv- 
orous animals.  Among  these,  the  sheep  and  goat  are  especially 
noteworthy,  as  in  these,  like  the  pure  carnivora,  taurocholic  acid 
only  is  found.  It  is  formed  synthetically  in  the  liver  from  taurin 
and  cholalic  acid,  and  is  accordingly  resolved  into  its  components 
by  treating  with  dilute  acids  or  alkalies  under  the  application  of 
heat.  The  same  result  indeed  is  reached  by  evaporating  the  bile 
together  with  water  or  allowing  it  to  undergo  putrefaction.  The 
chemical  change  is  represented  by  the  equation  : 

C,eH,,NSO,  +  H,0  =  C,H,NS03  +  C^.H^oOs 

Taurocholic  Taurin.  Cholalic 

acid.  acid. 

Tauber  succeeded  in  effecting  the  synthesis  of  taurin  and  cholalic 
acid  by  using  sodium  cholate,  while  with  the  free  acid  directly  no 
result  was  obtained.  Pellizari  and  Matteucci  arrived  at  similar 
results  by  starting  with  sodium-taurin  instead  of  free  taurin. 

In  the  pure  state  taurocholic  acid  occurs  in  the  form  of  fine  deli- 
quescent needles,  which  are  soluble  in  Avater  and  alcohol,  but 
insoluble  in  ether.  It  is  capable  of  maintaining  glycocholic  acid  in 
solution,  and  it  is  for  this  reason  that  the  latter  acid  cannot  be  pre- 
cipitated from  the  bile  by  adding  a  mineral  acid  when  taurocholic 
acid  is  at  the  same  time  present  in  sufficient  amount.  Like  glyco- 
cholic acid,  it  is  a  monobasic  acid,  and  forms  salts  which  for  the 
most  part  are  soluble  in  water  and  in  alcohol.  Its  salts  with  the 
alkalies  are  precipitated  from  their  solutions  by  subacetate  of  lead 
and  ammoniacal  subacetate  of  lead,  but  not  by  the  neutral  acetate,  by 
copper  sulphate,  or  silver  nitrate.  Curiously  enough,  the  sodium 
salt  obtained  from  the  bile  of  the  ox  is  dextrorotatory,  while  the 
same  salt  which  is  found  in  that  of  the  dog  turns  the  jilane  of 
polarization  to  the  left.  An  isomerism  thus  apparently  exists  which 
is  analogous  to  that  observed  in  the  case  of  the  tartaric  acids. 

Its  aqueous  solution  precipitates  albumins  in  large  white  flakes. 
With  albumose  solution  a  milky  precipitate  is  obtained  which  is 
largely  soluble  in  water  (Maly  and  Emich). 

Taurocholic  acid  forms  emulsions  with  the  peptones,  but  does  not 
precipitate  them,  as  is  generally  stated  ;  but  it  does  precipitate  albu- 
mins, syntonins,  and  albumoses. 

Hyotaurochofic  acid  is  the  biliary  acid  which,  as  a  sodium  salt,  is 
found  in  the  bile  of  pigs,  and  is  analogous  to  the  hyoglycocholic  acid 
already  described.  Its  amount,  however,  is  small.  On  decomposi- 
tion it  yields  taurin  and  hyocholalic  acid,  as  shown  in  the  equation  : 

a^H.jNSOfi   +   HP  =  C,5H,o04   +   C2H7NSO3 

Ilyotaurocholic  Hyocholalic  Taurin. 

acid.  acid. 

Chenotaurochollc  acid  is  the  most  im])ortant  biliary  acid,  Avhich 
is  found  in  the  bile  of  geese.     It  is  indistinctly  crystalline,  and  is 


THE  BILE.  167 

soluble  in  water  and  alcohol.     Like  the  acids  already  described,  it 

gives  Pettenkofer's  reaction.     On  prolonged   boiling  with  alkalies 

it  is  decomposed  into  taurin  and  chenocholalic  acid,  as  shown  in  the 

equation  : 

C^sH.sNSOfi   4-   HjO   =   C„H,,0,   +    aH,NS03 
Chenotaurocholic  Chenochola-  Taurin. 

acid.  lie  acid. 

Isolation. — Tanrocholic  acid  is  most  conveniently  obtained  from 
Platner's  bile  of  man  or  the  ox,  as  already  described.  To  isolate 
it  from  the  bile  of  dop-s,  the  fluid  is  shaken  with  animal  charcoal 
and  alcohol,  then  decanted  and  filtered.  The  filtrate  is  evaporated 
to  dryness,  the  residue  taken  up  with  a  small  amount  of  warm 
absolute  alcohol,  and  filtered.  The  clear  solution  is  then  treated 
with  ether  until  it  becomes  cloudy.  On  standing,  the  taurocholate 
of  sodium  separates  out  in  the  form  of  fine  crystals.  These  are 
dissolved  in  water  and  treated  with  ammoniacal  subacetate  of  lead. 
The  resulting  precipitate  is  suspended  in  alcohol  and  decomposed 
with  hydrogen  sulphide.  The  free  acid  is  thus  liberated,  and  can 
be  obtained  as  such  by  evaporating  the  filtrate  to  dryness  or  by 
diluting  with  ether. 

To  isolate  chenotaurocholic  acid,  the  bile  of  geese  is  first  treated 
with  strong  alcohol,  to  remove  the  mucus.  The  filtrate  is  mixed 
with  ether  and  set  aside,  when  the  biliary  salts  separate  out  as 
a  glutinous  mass,  which  is  then  washed,  dried,  redissolved  in  99  per 
cent,  alcohol,  and  again  precipitated  with  ether.  The  crystalline 
deposit  which  is  formed  is  dissolved  in  strong  alcohol  and  decom- 
posed with  hydrogen  sulphide.  On  evaporation  the  free  acid  is 
obtained. 

Cholalic  Acid. — Cholalic  acid  is  the  principal  biliary  acid  which 
is  fi)rmed  in  the  liver,  and  to  its  presence  in  the  molecule  of  glyco- 
cholic  acid  and  taurocholic  acid  Pettenkofer's  reaction  is  due.  It  is 
a  product  of  the  specific  activity  of  the  liver-cells,  and  is  normally 
not  found  in  any  other  tissues  or  organs  of  the  animal  body.  In 
the  intestinal  contents,  however,  it  may  be  encountered,  as  such, 
even  in  health,  and  in  cases  of  jaundice  it  has  been  observed  also  in 
the  ui-ine.  Its  artificial  introduction  in  dogs  leads  to  an  increased 
elimination  of  taurocholic  acid.  This  is  temporary,  and  ceases  when 
the  taurin,  which  has  been  stored  in  the  liver,  is  exhausted. 

As  I  have  already  shown,  cholalic  acid  is  liberated  when  glyco- 
cholic  acid  or  taurocholic  acid  is  decomposed  with  alkalies  or  acids, 
under  the  application  of  heat.  Its  crystalline  form  differs  accord- 
ing to  the  medium  in  which  crystallization  has  taken  place.  From 
its  alcoholic  solution  it  separates  out  in  the  form  of  rhqmbic 
tetrahedra  or  octahedra,  which  contain  one  molecule  of  alcohol  as 
liquid  of  crystallization,  Co^H^gO.,  +  C2II5O.  On  prolonged  boiling 
with  water  the  crystal-alcohol  can  be  removed.  When  dissolved  in 
dilute  boiling  acetic  acid  cholalic  acid  takes  up  one  molecule  of 
water,  and  can   be  obtained   from   such  solutions   in  the  form  of 


168  THE  DIGESTIVE  FLUIDS. 

rhombic  plates  or  prisms,  Co^H^oO^.HoO.  On  exposure  to  the  air 
the  crystals  in  either  case  soon  become  opaque.  Thev  melt  at  a 
temperature  of  ]9o°  C.  The  free  acid  is  readily  soluble  in  alcohol) 
with  difficulty  so  in  water,  and  is  almost  insoluble  in  ether. 

According  to  Mylius,  it  is  a  monobasic  alcoholic  acid,  and  contains 
one  secondarv  and  two  ])rimarv  alcohol  groups,  as  represented  by 

.CH.OH 
the  formula  :  C.,oH3j^(CH,.OH)o.     It  combines  with  alkalies  and 

COOH 
alkaline  earths,  as  also  with  the  heavy  metals,  to  form  salts.  Its 
compounds  with  the  alkalies  are  readily  soluble  in  water,  but  with 
some  difficulty  in  alcohol.  The  barium  salt  is  somewhat  soluble  in 
water,  and,  like  the  lead  salt,  soluble  in  hot  alcohol.  The  calcium 
salt  is  slightly  soluble  in  boiling  alcohol.  Concentrated  solutions  of 
the  alkaline  hydrates  and  carbonates  precipitate  the  alkaline  salts 
from  their  solutions  in  the  form  of  an  oily  material  which  becomes 
crystalline  on  cooling.  The  salts,  like  the  free  acid,  are  dextro- 
rotatory. 

On  prolonged  boiling  with  acids  or  at  a  temperature  of  200°  C. 
cholalic  acid  loses  two  molecules  of  water  and  is  transformed  into 
dyslysin,  as  shown  in  the  equation  : 

Cholalic         Dj-slysin. 
acid. 

The  same  result  is  brought  about  through  the  influence  of  various 
bacteria,  and  there  can  be  no  doubt  that  the  dyslysin  which  is  con- 
stantly encountered  in  the  stools  is  referable  to  the  normal  processes 
of  putrefaction,  which  take  place  in  the  intestinal  canal. 

Choloidinic  acid,  Co4H,^304,  probably  represents  an  intermediary 
product  whicli  is  formed  during  this  process,  and  may  be  regarded 
as  a  primary  anhydride  of  cholalic  acid. 

The  common  dyslysin  which  is  met  Avith  in  the  feces  is  amorphous, 
and  is  insoluble  in  water  and  in  dilute  solutions  of  the  alkalies. 

On  oxidation  with  permanganate  cholalic  acid  first  yields  dehydro- 
cholaliG  acid,  and  then  bilianic  acid,  together  with  isobilianic  acid. 
These  changes  may  be  represented  by  the  equations  : 

(1)  C,,H«0,    +    30    =    C,,H,A    -    3H,0 
Cholalic  Dehydrocholalic 

acid.  '  acid. 

(2)  C,,H3,05     -r     30    =    C.HjPe 
Dehydrochol-  Bilianic 

al'ic  acid.  acid. 

On  further  oxidation  another  acid  has  been  obtained,  which  is 
termed  cilianic  acid,  C2„H.,gOg. 

Dehydrocholalic  acid  also  results  on  oxidizing  cholalic  acid  with 
nitric  acid  ;  but  it  is  to  be  noted  that  on  further  oxidation  a  new 
acid  results,  which  is  known  as  choleocamphoric  acid,  and  is  thought 
to  be  isomeric  with  camphoric  acid.  Its  formula  is  given  as  Ci^Hi^O^. 
On  oxidation  with  potassium  bichromate  and  sulphuric  acid,  on  the 


THE  BILE.  1G9 

other  hand,  Tujipeiner  claims  to  liave  obtained  cholesteric  acid  (not 
to  be  confonnded  with  cliolesterinic  acid,  see  below),  C12H10O7  ', 
pyrocholesteric  acid,  Ci,HiyO- ;  cholanic  acid,  CjoHggOg;  as  also 
palmitic,  stearic,  and  acetic  acids. 

On  rednction,  as  dnring  the  process  of  putrefaction,  cholalic  acid 
may  give  rise  to  the  formation  of  dcsoxf/cho/alic  acid,  C24H^oC^4'  C)n 
boiling  with  concentrated  solutions  of  the  alkaline  hydrates  a  mixt- 
ure of  the  corresponding-  salts  of  formic  acid,  acetic  acid,  propionic 
acid,  and  palmitic  acid  is  obtained,  and  it  is  interesting  to  note  that 
the  latter,  like  cholalic  acid,  gives  Pettenkofer's  reaction. 

Flyocholalic  acid  and  chenocholalic  acid,  which  result  from  the 
decomposition  of  hyotaurocholic  acid,  hyoglycocholic  acid,  and 
chenotaurocholic  acid,  respectively,  in  their  general  properties  and 
reactions  are  closely  analogous  to  the  common  form  of  cholalic  acid 
that  has  just  been  considered.  Like  the  latter,  they  are  trans- 
formed in  the  intestinal  tract  into  the  corresponding  dyslysins, 
and  tiiese  in  turn  yield  the  original  acids  on  heating  with  sodium 
hydrate  solution.  Both  are  soluble  in  alcohol  and  ether,  but 
insoluble  in  water. 

Isolation  of  Cholalic  Acid  from  the  Bile. — Platner's  bile,  olitained 
from  the  ox,  is  boiled  for  twenty-four  hours  with  barium  hydrate. 
The  cholalic  acid  which  is  thus  set  free  is  precipitated  by  adding 
an  excess  of  hydrochloric  acid.  It  is  then  washed  with  water, 
dissolved  in  a  dilute  solution  of  sodium  carbonate,  and  repre- 
cipitated  with  hydrochloric  acid.  The  precipitate  is  covered  Avith 
ether  and  allowed  to  stand,  when  crystallization  will  gradually  occur. 
The  crystals  are  freed  from  liquid  as  far  as  possible  by  filtration  with 
the  suction-pump,  and  are  redissolved  in  hot  alcohol.  The  solution 
is  diluted  with  water  until  a  persisting  turbidity  develops,  and  is 
then  set  aside  in  a  cold  place  until  crystallization  is  complete. 

Choleic  Acid. — When  the  biliary  acids  of  ox-bile  are  decomposed, 
as  just  described,  a  small  amount  of  choleic  acid,  C^^^^f}^,  is  always 
found  associated  with  common  cholalic  acid.  On  oxidation  it  first 
yields  dehydroclioleic  acid,  G.,Ji■^^0^,  and  then  cholanic  acid,  C24H340g. 
It  is  possibly  identical  with  desoxycholalic  acid  (see  above).  The 
substance  has  also  been  found  in  human  bile. 

Fellic  Acid. — This  acid  has  been  obtained  together  with  common 
cholalic  acid  and  choleic  acid  from  human  bile,  where  it  possibly 
exists  in  combination  with  glycocoU  and  taurin.  Its  amount  is 
always  small.  With  Pettenkofer's  test  it'  gives  a  reddish-blue 
color.  On  heating,  it  is  decomposed,  with  the  formation  of  vapors 
which  are  strongly  suggestive  of  turpentine.  Its  formula  is  given 
as  C^sH^O,. 

Lithofellic  Acid. — Lithofellic  acid  is  a  substance  which  is  closely 
related  to  the  cholalic  acids  just  described,  and  is  found  in  certain 
gastro-intestinal  concretions  of  various  ruminants.  It  forms  the 
greater  portion  of  the  oriental  Bezaar  stones,   which    are  obtained 


170  THE  DIGESTIVE  FLUIDS. 

from  the  stomach  of  the  wild  goat  aiul  antelope.  It  gives  Petten- 
kofer's  reaction,  and  is  said  to  have  the  forninla  G,nHj|.,0^. 

Taurin. — As  has  been  pointed  out,  taurin  is  not  foinid  exclusively 
in  the  bile,  but  occurs  also  in  the  lungs,  kidneys,  and  in  the  muscle- 
tissue  of  many  vertebrate  animals.  ]t  is  a  derivative  of  cystin, 
and  thus  of  albuminous  origin.  It  is  noteworthy,  however,  that  an 
increased  ingestion  of  albumins  to  eight  times  the  usual  amount  in- 
creases the  biliary  sulphur  (taurocholic  acid)  only  twice.  This  finds 
its  explanation  in  the  observation  of  v.  Bergmann  that  cystin  jwr  se 
will  not  lead  to  an  increased  elimination  of  the  bile  sulphur,  when  fed 
directly,  while  this  occurs  when  cholalic  acid  is  simultaneously  ad- 
ministered. We  may  accordingly  conclude  that  under  usual  condi- 
tions a  sufficiently  large  amount  of  cholalic  acid  is  not  available  for 
the  elimination  of  a  larger  amount  of  taurin  than  ordinarv  as  tauro- 
cholic  acid.  There  is  evidence  that  in  dogs  there  is  a  store  of  taurin 
(or  of  cystin)  normally,  which  rapidly  disappears  when  cholalic  acid 
is  administered,  as  evidenced  by  an  increased  output  of  the  bile  sul- 
phur. Personal  investigations  in  association  with  Dr.  D.  G.  Camp- 
bell have  led  me  to  similar  results  in  man. 

Of  the  manner  in  which  cystin  gives  rise  to  taurin  our  knowledge 
is  not  yet  complete.  Different  possibilities  exist.  On  the  one  hand, 
the  cystin  may  be  oxidized  to  cystei'nic  acid,  which  apparently 
represents  the  sulpho-acid  of  cystein,  and  from  which  taurin  could 
then  result  through  loss  of  CO2  as  expressed  by  the  equations : 


CH,.S— S.CH2 

CH2.SO3H 

CH.NH»  CH.NH, 

1                 1 

+  50  +  Hfi  =  2CH.NH2 

COOH       COOH 

Cystin 

COOH 
Cyste'inic  acid 

CH^.SOsH 

1 
CH.NH^              = 

COOH 

Cyste'inic  acid 

CH2.SO3H 

1                   +       CO, 
CHj.NHj 

Taurin 

It  is  possible,  on  the  other  hand,  that  the  organism  forms  taurin 
directly  by  oxidation  of  the  thio-  to  the  sulj)ho-group  with  the  co- 
incident loss  of  carbon  dioxide. 

Then  again  there  is  a  possibility  that  cystin  may  unite  with  chola- 
lic acid  primarily,  and  that  the  resulting  product  is  subsequently 
oxidized  to  taurocholic  acid  without  the  intermediate  formation  of 
taurin. 

Unlike  the  loosely  combined  sulphur  of  cystin,  the  sulphur  of 
taurin  cannot  be  split  off  on  boiling  with  dilute  alkalies.  It  is 
present  in  oxidized  form.  Its  separation  necessitates  the  complete 
destruction  of  the  taurin. 

Wahlgreen    has   recently  isolated   the   corresponding  glycocholic 


THE  BILE.  171 

acid  from  ox-bile,  and  it  is  possible  that  this  is  identical  with  Mul- 
der's eholonic  acid. 

In  the  bile  of  ice-bears  Haramarsten  has  found  a  cholei'c  acid  of 
the  composition  Ci9H3i,04  or  CigH2g04 ,  which  is  thus  homologous  to 
the  common  choleic  ;  he  terms  it  ursocholeic  acid. 

Taurin  can  be  formed  synthetically  by  heating  ammonium-oxy- 
ethyl-sulplionate  to  a  temperature  of  230°  C,  or  from  ammonia  and 
chlor-ethyl-sulphonic  acid,  as  rej^resented  by  the  equations : 

NH3  +  SOj<  =  so/  '  +  HCl 

\0H  \0H 

Chlor-ethyl-sul-  Taurin. 

phonic  acid. 

/CjH^.OH  .C2H,.NH2 

so/  =S0/  +H5O 

\O.NH,  \0H 

Ammonium  oxy-  Tauriu. 

ethyl-sulphonic 
acid. 

It  can  hence  be  regarded  as  amido-ethyl-sulphonic  acid  (viz., 
amido-isethionic  acid).  It  crystallizes  in  the  form  of  four-  or  six- 
sided  prisms,  and  is  fairly  soluble  in  hot  water,  slightly  soluble  in 
common  alcohol,  and  insoluble  in  absolute  alcohol  and  ether.  It 
has  a  markedly  acid  character,  and  accordingly  does  not  combine 
with  acids,  but  with  alkalies,  to  form  sails.  Its  compound  with 
mercuric  oxide  is  quite  insoluble. 

Isolation  of  Taurin. — Taurin  is  most  conveniently  prepared  from 
the  bile  of  animals  in  which  taurocholic  acid  is  present.  To  this 
end,  the  fluid  is  boiled  for  several  hours  with  hydrochloric  acid. 
The  dysly^sin  and  choloidinic  acid  are  filtered  off.  The  filtrate  is 
concentrated  on  a  water-bath  to  a  small  volume,  and  freed  from  the 
sodium  chloride  and  other  substances  that  have  separated  out,  by 
filtering  while  still  warm.  The  liquid  is  then  evaporated  to  dryness 
and  the  residue  extracted  with  strong  alcohol,  which  dissolves  any 
glycocoll  hydrochlorate  that  may  be  present,  while  the  taurin  re- 
mains behind.  This  is  then  dissolved  with  a  little  warm  water,  fil- 
tered while  still  warm,  and  treated  with  an  excess  of  warm  alcohol. 
The  resulting  precipitate  is  immediately  filtered  oflP.  In  the  filtrate 
the  taurin  crystallizes  out  on  cooling, and  can  be  identified  by  the  form 
of  its  crvstals,  their  solubilitv  in  water  and  insolubility  in  alcohol, 
and  the  formation  of  potassium  sulphate  when  fused  with  potassium 
hydrate  and  potassium  nitrate. 

Should  glycocholic  acid  be  present  in  the  bile  at  the  same  time, 
this  is  likewise  decomposed  on  boiling  with  hydrochloric  acid.  The 
hydrochlorate  of  glycocoll  which  thus  results  is  found  in  the  first 
alcoholic  extract.  To  isolate  the  glycocoll  as  such,  the  solution  is 
evaporated  to  dryness,  the  residue  dissolved  in  water  and  treated 
with  plumbic  hydrate.  On  filtering,  the  solution,  which  contains 
the  lead  salt  of  glycocoll,  is  decomposed  with  hydrogen  sulphide. 
The  resulting  lead  sulphide   is  filtered  off,  and  the  filtrate  concen- 


172  THE  DIGESTIVE  FLUIDS. 

trateJ  until  crystallization  occurs.  The  crystals  are  then  dissolved 
in  water  and  decolorized  with  animal  charcoal,  and  the  solution  is 
evaporated  until  the  crystals  again  separate  out. 

Glycocoll. — GlycocoU  is  now  known  to  be  a  constant  decomposi- 
tion-product of  most  albumins,  but  is  formed  in  especially  large 
amoiuits  during  the  hydrolytic  decomposition  of  collagen  and 
spongin.  From  this  fact  and  its  sweetish  taste  it  is  also  known 
as  glucin  or  collagen-sugar  (Leimzucker).  It  is  one  of  the  most 
important  decomposition-products  which  are  formed  in  the  nitro- 
genous metabolism  of  the  animal  body,  and  in  part  at  least  gives 
rise  to  the  formation  of  urea  in  mammals,  and  to  uric  acid  in  birds 
and  reptiles.  Another  portion,  as  we  have  seen,  is  eliminated  in 
the  bile  in  combination  with  cholalic  acid ;  while  a  third  portion 
appears  in  the  urine  in  combination  with  benzoic  acid  and  phenyl- 
acetic  acid,  as  hippuric  acid  and  phenaceturic  acid,  respectively  (which 
see). 

As  we  have  seen,  glycocoll  is  amido-acetic  acid.  The  pure  sub- 
stance crystallizes  in  the  form  of  colorless  rhorabohedra  or  of  four- 
sided  prisms,  which  are  readily  soluble  in  water,  with  difficulty  so 
in  warm  alcohol,  and  insoluble  in  absolute  alcohol  and  ether.  It 
combines  with  acids  and  alkalies  to  form  salts.  The  most  important 
of  these  are  the  hydrochlorate,  which  is  soluble  in  water  and  alcohol, 
and  the  copper  salt,  which  results  when  a  boiling  solution  of  glycocoll 
is  added  to  freshly  precipitated  cupric  hydrate;  the  hydrate  is  thus 
dissolved,  and  after  concentrating  the  solution  blue  needles  of  the 
copper  salt  sejiarate  out  on  cooling. 

Isolation. — Glycocoll  can  be  obtained  from  the  bile  of  those 
animals  in  which  glycocholic  acid  is  found,  as  described  above ;  or 
it  may  be  prepared  from  hippuric  acid  by  decomposing  this  by  boil- 
ing with  dilute  sulphuric  acid.  On  cooling,  the  benzoic  acid  that 
has  separated  out  is  filtered  off,  the  filtrate  is  concentrated  and  ex- 
tracted with  ether  to  remove  such  benzoic  acid  as  still  remains  in 
solution;  the  sulphuric  acid  is  removed  with  barium  carbonate,  and 
the  filtrate  is  evaporated  until  crystals  of  glycocoll  begin  to  separate 
out  (see  also  pages  210  and  278). 

The   Bile -pigments. 

The  bile-pigments  which  have  thus  far  been  obtained  from  the 
bile  itself  or  from  biliary  concretions  are  bilirubin,  biliverdin,  bili- 
prasin,  bilifuscin,  and  others  which  are  less  well  known. 

Perfectly  fresh  hepatic  bile,  in  contradistinction  to  that  which  is 
found  in  the  gall-bladder,  contains  only  one  pigment,  bilirubin,  from 
which  all  other  forms  are  derived.  Such  bile  is  of  a  golden-yellow 
color,  while  bladder-bile  usually  presents  an  olive-brown  color,  owing 
to  the  simultaneous  presence  of  its  nearest  oxidation-product,  bili- 
verdin. A  grass-green  color  is  observed  when  the  latter  predomi- 
nates or  is  exclusively  present. 


THE  BILE.  173 

Bilirubin. — Bilirubin  is  now  known  to  result  from  the  decompo- 
sition of  haematin,  and  normally  constitutes  a  specific  product  of  the 
activity  of  the  hepatic  cells.  It  appears,  however,  that  the  power 
of  transforming  the  blood-pigment  into  bilirubin  is  common  to  other 
tissues  as  well,  if  we  regard  the  hsematoidin  of  Virchow,  which  is 
so  often  found  in  old  extravasations  of  blood,  as  identical  with  bili- 
rubin. That  this  is  actually  the  case  seems  now  undoubted.  Under 
normal  conditions,  however,  the  liver  is  apparently  the  only  organ 
of  the  body  in  wllich  the  formation  of  bilirubin  takes  place. 
Whether  or  not  the  final  dissolution  of  disintegrating  red  corpuscles 
also  occurs  at  this  place  has  not  been  decided,  but  the  liver  is 
manifestly  capable  of  retaining  the  haemoglobin  which  is  thus  set 
free.  If  a  moderate  amount  of  a  solution  of  haemoglobin  is  injected 
into  the  bloodvessels  of  an  animal,  an  increased  elimination  of 
bilirubin  results,  Avhile  the  blood-pigment  does  not  appear  in  the 
urine.  If  larger  amounts  are  injected,  the  resulting  bilirubin  ap- 
parently cannot  be  removed  with  sufficient  rapidity  through  the 
biliary  ducts.  Resorption  of  the  pigment  then  takes  place  through 
the  lymph-vessels,  jaundice  results,  and  the  bile-pigment  appears  in 
the  urine.  If  excessive  amounts  of  haemoglobin  finally  are  injected, 
the  liver  is  manifestly  incapable  of  retaining  all  the  pigment  which 
reaches  the  organ,  jaundice  results,  and  both  the  bile-pigment  and 
the  blood-pigment  appear  in  the  urine.  Even  in  such  extreme 
cases  the  remaining  tissues  of  the  body  do  not  participate  in  the 
formation  of  bilirubin.  This  has  been  conclusively  demonstrated 
by  Minkowski  and  Naunyn.  These  observers  have  shown  that  while 
in  normal  geese  poisoning  with  hydrogen  sulphide,  which  causes 
an  extensive  dissolution  of  the  red  corpuscles,  invariably  leads  to 
jaundice  and  the  appearance  of  bile-pigment  in  the  urine,  the 
previous  removal  of  the  liver  prevents  such  an  occurrence,  and 
results  in  simple  hfemoglobinuria.  In  mammals  such  crucial  tests 
unfortunately  cannot  be  applied,  but  there  is  no  reason  to  suppose 
that  different  conditions  there  exist.  The  possible  occurrence  of  a 
haematogenic  icterus,  in  contradistinction  to  a  hepatogenic  icterus,  is 
thus  rendered  extremely  improbable,  and  it  is  scarcely  warrantable 
to  point  to  the  development  of  bilirubin  from  blood-pigment  in  the 
tissues  of  the  organs,  as  indicating  the  possibility  of  such  a  trans- 
formation in  the  circulating  blood. 

Of  the  manner  in  which  this  transformation  is  effected  m  the  liver 
we  know  nothing.  We  may  imagine,  however,  that  the  oxyhaemo- 
globin  is  here  first  decomposed  into  its  albuminous  component — 
globin — and  into  haematin,  which  latter  then  passes  over  into  bili- 
rubin, as  shown  in  the  equation  : 

C3,H3,N  AFe  +  2H,0  -  Fe  =  C,,ll,,^,0, 
Htematin.  Bilirubin. 

This  reaction,  it  will  be  noted,  is  also  supposed  to  express  the 
formation  of  haematoporphyrin  from  haematin  (see  page  357).     The 


174  THE  DIGESTIVE  FLUIDS. 

two  substances,  indeed,  are  now  quite  generally  regarded  as  isomeric. 
As  regards  the  size  of  the  molecule,  however,  opinions  differ,  and  it 
is  possible,  as  Xencki  and  Sieber  suppose,  that  tiie  molecule  of  bili- 
rul)in,  as  well  as  of  hsematoporphyrin,  is  only  half  as  large  as 
expressed  above.  In  that  case,  of  course,  the  equation  would  have 
to  be  written  : 

CajHjjNAFe  ^  2H3O  -  Fe  =  2CieHi8N,03 

On  oxidation  bilirul)in  yields  the  same  hsematinic  acids  which 
Kiister  obtained  from  hiematin  and  hsematoporphyrin. 

On  reduction  with  nascent  hydrogen  Ijilirubin  is  transformed  into 
hydrobilirubin,  as  is  shown  by  the  equation  : 

Ca^HjeN.Osfviz,  2CieHi8N,03)  +  H,0  +  2H  =  Ca^H^X.O, 

Hydrobilirubin. 

This  actually  takes  place  in  the  intestinal  canal,  where  nascent 
hydrogen  is  constantly  being  formed  through  the  action  of  various 
bacteria,  as  during  the  process  of  lactic  acid  fermentation.  During 
intra-uterine  life,  however,  where  no  bacteria  are  found  in  tlie 
intestinal  tract,  bilirubin  appears  in  the  feces  as  such  (meconium). 

According  to  some  observers,  hydrobilirubin  is  thought  to  be 
identical  with  the  febrile  urobilin  of  Jaffe,  which  is  met  ^vith  in  the 
urine  in  various  febrile  diseases,  and  exceptionally  also  under  normal 
conditions,  but  it  is  said  to  differ  from  the  normal  urobilin  which 
represents  the  principal  coloring-matter  of  the  urine  in  health. 
This  question,  however,  still  awaits  solution.  The  possible  identity 
also  of  hydrobilirubin  and  stercobilin,  which  is  the  principal  pig- 
ment that  is  found  in  the  feces,  has  not  been  definitely  established 
(see  also  pages  225  and  314). 

In  the  crystalline  state  bilirubin  occurs  in  the  form  of  reddish- 
yellow  rhombic  platelets  with  rounded  angles,  which  are  solul)le  in 
benzol,  carbon  disulphide,  arayl  alcohol,  glycerin,  the  fatty  oils,  and 
especially  in  warm  chloroform.  In  alcohol  and  ether  they  are  but 
little  soluble,  and  in  water,  as  also  in  Platner's  bile,  they  are  insol- 
uble. On  spectroscopic  examination  its  solutions  do  not  give  rise  to 
any  specific  bands  of  absorption,  but  to  a  continuous  absorption 
which  extends  from  the  red  to  the  violet  end. 

Bilirubin  as  such  is  a  weak  acid,  bUirubinic  acid,  and  combines 
with  bases  to  form  salts,  which  for  the  most  part  are  either  insoluble 
or  only  slightly  soluble  in  water  and  insoluble  in  chloroform.  Its 
salts  with  the  alkalies,  however,  are  soluble  in  solutions  of  the  alka- 
line hydrates  and  carbonates.  In  the  bile  bilirubin  is  largely  present 
as  neutral  bilirubinate  of  sodium,  and  is  held  in  solution  owing  to 
the  presence  of  alkaline  carbonates. 

When  perfectly  fresh  bile  of  a  golden-yellow  or  olive-brown  color 
is  exposed  to  the  air  for  a  while,  it  will  be  noted  that- the  fluid  grad- 
ually assumes  a  bright-green  color,  owing  to  a  transformation  of  the 
bilirubinate  into  bilivercUnate  of  sodium.     Free  bilirubin,  according 


THE  BILE.  175 

to  Dastre  antl  Floresco,  does  not  absorb  oxygen  and  is  thereby 
transformed  into  biliverdin,  as  has  been  snpposed.  The  same 
observers  state  that  on  careful  oxidation  biHrubin  can  be  trans- 
formed into  biliprasin,  which  presents  a  green  color  as  such,  while 
its  sodium  salt,  sodium  hUiprasinate,  is  yellowish  brown.  On 
further  oxidation  the  biliprasin  then  yields  biliverdin,  viz.,  its  cor- 
responding salt. 

According  to  former  views,  the  relation  existing  between  bilirubin 
and  biliprasin  were  represented  by  the  equations  given  below ;  but 
it  is,  of  course,  manifest  that  these  are  no  longer  tenable  if  the  work 
of  Dastre  and  Floresco  should  be  confirmed. 

C,eH,8N,03  +  0  =  C,eH,8N,0, 

Bilirubin.  Biliverdin. 

Cielf.sNA  +  H,0  =  C,«H,oNA 

Bilirubin.  Bilifuscin. 

C,fiH,„N20,  +  HP  +  O  =  C,«H,,N  A 
Bilifuscin.  Biliprasin. 

The  formula  of  biliprasin,  as  here  indicated,  would,  moreover,  be 
an  impossibility.  As  a  matter  of  fact,  these  formulae  cannot  be 
regarded  as  definitely  established ;  and  according  to  some  observers, 
the  molecule  of  biliverdin  is  only  one-half  as  large  as  represented 
above.  Much  work  still  remains  to  be  done  in  this  connection,  but 
we  know  at  least  that  biliverdin  constitutes  a  normal  oxidation- 
product  of  bilirubin.  From  biliverdin  Kiister  claims  to  have 
obtained  a  new  oxidation-product,  which  he  termed  hiliverdinic  acid, 
but  which  must  not  be  confounded  with  the  substance  of  the  same 
name  referred  to  above.  This  body  has  the  formula  CgH^NO^,  and 
is  apparently  identical  with  the  dibasic  ha^matinic  acid,  which  results 
from  haematin  directly,  and,  like  this,  yields  a  substance  of  the  com- 
position CgHgO^  viz.,  the  anhydride  of  the  dibasic  haematinic  acid 
CgHj|,Og.  In  this  manner  the  origin  of  bilirubin  from  the  coloring- 
matter  of  the  blood  is  still  further  shown. 

If  bile  containing  bilirubin  is  filtered  through  Swedish  filter- 
paper,  and  a  drop  of  concentrated  nitric  acid  containing  a  trace 
of  nitrous  acid  is  placed  upon  the  paper,  which  is  colored  a  bright 
yellow,  a  play  of  colors  will  be  observed,  in  which  the  yellow  first 
turns  to  green,  then  to  blue,  to  violet,  to  red,  and  ultimately  to 
orange.  This  reaction  is  commonly  referred  to  the  formation  of 
various  oxidation-products  of  bilirubin,  and  is  very  characteristic. 
The  individual  products,  however,  which  thus  result  are  mostly  Init 
imperfectly  known.  The  green  color,  of  course,  is  referable  to  the 
biliverdin.  The  blue  color  is  ascribed  to  bilicyanin  or  cholecyanin, 
and  the  final  orange  to  choletelin. 

Tests  for  Bilirubin. — Gmelin's  Test. — The  fluid  to  be  examined 
is  treated  with  an  amount  of  concentrated  nitric  acid,  containing  a 
trace  of  nitrous  acid,  sufficient  to  form  a  layer  beneath  the  liquid  to 
be  tested,  when  in  the  presence  of  bilirubin  tiie  color-play  referred  to 
will  be  observed  at  the  zone  of  contact ;  the  green  will  be  noticed 


176  THE  DIGESTIVE  FLUIDS. 

nearest  the  bile-containiug  solution,  and  the  orange  in  the  upper 
portion  of  the  nitric  acid.  Various  modifications  of  this  reaction 
have  been  proijosed,  such  as  the  one  described  above. 

The  test  is  exceedingly  sensitive,  and  is  said  to  indicate  the  pres- 
ence of  bilirubin  iu  a  dilution  of  1  :  80,000.  The  green  color  which 
develops  is  the  most  characteristic,  but  a  reddish  violet  must  also 
occur. 

Huppert's  Test. — A  few  cubic  centimeters  of  the  solution  to 
be  examined  are  precipitated  with  barium  chloride  and  ammonia. 
The  precipitate  is  washed  with  water  and  suspended  in  a  small 
amount  of  alcohol  that  has  been  acidulated  with  sulpliuric  acid. 
This  mixture  is  then  boiled  for  a  few  minutes,  when  in  the  presence 
of  bilirubin  a  bi'ight  emerald-green   color  develops. 

Smith's  Tfjst. — A  small  amount  of  the  fluid  is  placed  in  a  test- 
tube  and  treated  with  a  few  cubic  centimeters  of  tincture  of  iodine 
which  has  been  diluted  with  alcohol  in  the  proportion  of  1  :  10,  so 
as  to  form  a  layer  above  the  fluid  to  be  examined.  In  the  presence 
of  bilirubin  a  distinct  emerald-green  ring  will  develop  at  the  zone 
of  contact. 

According  to  some  observers,  the  green  color  which  thus  results 
when  bilirubin  is  treated  with  iodine  is  not  referable  to  the  forma- 
tion of  biliverdin,  but  to  a  substitution-  or  addition-product  of  bili- 
verdin  with  iodine.  This,  however,  is  denied  by  otiiers,  and  Jolles 
has  recently  shown  that  the  iodine  merely  acts  as  an  oxidizing  agent, 
and  that  true  biliverdin  is  thus  formed,  as  indicated  by  the  equation  : 

CieHigN^Os  -  21  -  H^O  =  CieHigNA  ^  2HI. 

Spectroscopic  Test. — If  a  dilute  solution  of  sodium  bilirul)i- 
nate  in  water  is  treated  with  an  excess  of  ammonia  and  a  small 
amount  of  a  solution  of  chloride  of  zinc,  the  liquid  at  first  turns 
a  deep  orange,  but  subsequently  becomes  olive  brown,  and  finallv 
green.  If  this  solution  is  examined  spectroscopically,  it  will  be 
noted  that  the  violet  and  blue  portions  of  the  spectrum  are  at  first 
quite  dark,  but  subsequently  the  bands  presented  by  an  alkaline 
solution  of  bilicyanin  become  apparent,  and  notably  the  one  between 
C  and  D,  near  C  (see  below).  The  test  is  said  to  be  very  good 
(Hammarsten). 

Isolation  of  Bilirubin. — Bilirubin  is  most  convenientlv  obtained 
from  the  biliary  concretions  which  are  so  often  found  in  the  gall- 
bladder of  cattle,  and  which  consist  almost  entirely  of  the  calcium 
salt  of  the  pigment.  They  are  finely  powdered  and  extracted  with 
ether  and  then  A^^th  hot  water,  so  as  to  remove  the  cholesterin  and 
the  biliary  acids  wiiich  are  present.  The  remaining  material  is 
treated  with  hydrochloric  acid,  so  as  to  liberate  the  pigment.  It  is 
then  washed  free  from  acid  with  water,  and  subsequently  with  abso- 
lute alcohol  to  remove  the  water  and  any  biliverdin  that  raav  be 
present.  The  pigment  remains,  and  is  now  dissolved  with  boiling 
chloroform.     From  this  solution  the  chloroform  is  distilled  ofi\,  the 


THE  BILE.  Ill 

residue  extracted  with  al)S(jliite  alcohol,  so  as  to  remove  any  bili- 
fiiscin,  and  the  remaining  bilirubin  dissolved  in  a  small  amount  of 
chloroform  and  precipitated  with  alcohol.  This  procedure  is 
repeated  until  the  substance  has  been  obtained  in  pure  form  ;  it 
is  then  allowed  to  crystallize  out  from  its  chloroform  solution  on 
cooling. 

Biliverdin. — Biliverdin  is  found  in  the  bladder-bile  of  many  ani- 
mals together  with  bilirubin,  and  is  especially  abundant  in  certain 
herbivora,  where  the  bile  frequently  presents  a  bright  grass-green 
color.  It  is  said  to  occur  also  in  the  placenta  of  the  bitch,  in  the 
shells  of  certain  mollusks,  and  in  birds'  eggs.  Its  relation  to  bili- 
rubin has  already  been  considered.  In  the  bile  it  is  present  princi- 
pally in  the  form  of  its  sodium  salt,  and,  like  bilirubin,  the  free 
pigment  possesses  acid  properties ;  this  is  termed  biliverdinic  acid, 
but  should  not  be  confounded  with  the  acid  of  the  same  name 
which  Kiister  obtained  from  biliverdin  on  oxidation  with  sodium 
chromate.  In  acid  bile  biliverdin  is  found  as  such.  Unlike 
bilirubin,  the  free  pigment  is  readily  soluble  in  normal  as  well  as  in 
neutral  and  acid  bile.  It  is  insoluble  in  water,  ether,  and  chloro- 
form, but  dissolves  in  alcohol,  glacial  acetic  acid,  and  solutions  of 
the  alkalies.  From  the  latter  it  is  precipitated  by  the  salts  of  the 
alkaline  earths  and  the  heavy  metals,  as  also  by  acids.  On  treating 
an  alcoholic  solution  of  biliverdin  with  ammoniacal  chloride  of  zinc 
solution  the  fluid  exhibits  a  green  fluorescence.  The  green  color  of 
the  pigment  is  changed  to  yellow  if  its  solution  in  acid  alcohol  is 
treated  with  zinc.  If  chlorine- water  is  added  instead,  a  blue  color 
develops  at  the  bottom  of  the  liquid,  and  above  it  layers  present- 
ing a  violet,  a  red  and  a  yellow  color  will  be  observed.  On  adding 
an  excess  of  chlorine-water  the  solution  is  decolorized. 

Pure  biliverdin,  like  bilirubin,  gives  no  characteristic  band  of 
absorption  in  alkaline  solution.  In  acid  solution,  however,  or  in 
pure  alcoholic  solution,  an  indistinct  baud  is  observed  at  D,  and  one 
that  is  more  pronounced  near  F. 

The  substance  is  amorphous,  or  at  least  cannot  be  obtained  in  a 
pronounced  crystalline  form. 

On  reduction,  biliverdin  is  supposedly  transformed  into  bilirubin, 
though  this  is  denied  by  some  observers. 

On  oxidation  with  nitric  acid  biliverdin  gives  rise  to  the  various 
colors  which  have  already  been  described,  beginning  with  blue.  It 
gives  Huppert's  reaction  directly. 

Isolation. — To  prepare  biliverdin,  it  is  most  convenient  to  start 
with  a  solution  of  bilirubin  in  the  form  of  its  sodium  salt  which  has 
been  exposed  to  the  air  until  the  original  golden-yellow  color  has 
changed  to  a  brownish  green.  The  biliverdin  is  then  precipitated 
by  adding  an  excess  of  hydrotihloric  acid.  It  is  filtered  ofl",  washed 
free  from  all  acid,  dissolved  in  absolute  alcoiiol,  and  precipitated  by 
copiously  diluting  with  water.  Any  bilirubin  that  may  be  present 
is  removed  by  extracting  with  chloroform. 

12 


178  THE  DIGESTIVE  FLUIDS. 

Biliprasin. — Biliprasin  as  such,  and  biliprasiuate  of  sodium,  ac- 
cording to  Dastre  and  Floresco,  are  also  found  in  normal  bladder- 
bile,  and,  as  has  been  indicated,  represent  intermediary  products  of 
oxidation  between  bilirubin  and  biliverdin. 

The  sodium  salt,  according  to  these  observers,  is  a  yellowish-brown 
pigment,  and  can  be  transformed  into  biliprasin  through  the  agency 
of  mineral  acids,  of  acetic  acid,  and  even  of  carbonic  acid.  On  ex- 
posure to  the  air  it  passes  over  into  the  corresponding  biliverdinate. 
To  its  presence  the  yellow  color  of  the  bile  of  calves  and  of  other 
animals  is  supposedly  due. 

Biliprasin  itself  is  green,  but  can  be  transformed  into  the  yellow 
salt  by  adding  a  few  drops  of  an  alkali,  and  this  in  turn  yields  the 
green  pigment  on  treating  with  an  acid.  This  reaction,  according 
to  Dastre  and  Floresco,  explains  the  fact  that  yellow  bile  can  become 
green  without  oxidation,  viz.,  without  the  formation  of  biliverdin. 
According  to  older  ideas,  however,  biliprasin  is  an  oxidation-product 
of  biliverdin,  and  is  supposed  to  result  from  this  with  the  inter- 
mediary formation  of  bilifuscin,  as  has  already  been  outlined. 

Bilifuscin  is  said  to  occur  in  gall-stones  together  with  bilirubin. 
The  formula  which  has  been  ascribed  to  it  is  that  of  bilirubin  plus 
one  or  two  molecules  of  water,  viz.,  CojH^^X^O^  or  C^.H^oN^O^.  It 
is  of  a  dark-brown  color,  and  is  soluble  in  alcohol  and  the  solutions 
of  the  alkaline  hydrates,  but  insoluble  in  water  and  chloroform. 
In  pure  form  it  does  not  give  Gmelin's  reaction. 

Bilicyanin,  or  cholecyanin,  is  the  blue  pigment  which  is  formed 
during  the  oxidation  of  bilirubin  and  biliverdin  with  nitric  acid.  It 
has  been  found  together  with  the  common  l)ile-pigments  in  gall- 
stones taken  from  man.  Its  neutral  and  alkaline  solutions  give  rise 
to  three  bands  of  absorption.  One  of  these  is  located  between  C 
and  D,  near  C ;  another  about  D ;  and  a  third  very  faint  band 
midway  between  D  and  E.  In  acid  solutions  two  bands  are  seen 
between  C  and  E.  On  treating  the  alcoholic  solution  of  the  pig- 
ment with  an  ammoniacal  solution  of  zinc  chloride  a  distinct  fluor- 
escence is  obtained.  Its  formula  has  not  as  yet  been  determined, 
and,  according  to  some  observers,  indeed,  bUicyanin  does  not  repre- 
sent a  separate  substance. 

Bilipurpurin. — This  term  has  been  a])plied  to  a  red  pigment 
which  is  formed  from  bilirubin  and  biliverdin  on  treating  with 
nitric  acid.  A  pigment  of  the  same  name  has  been  isolated  from 
ox-gall  by  Lobisch  and  Fischler.  Its  formula  is  given  as  C^^Hg^- 
N,0- ,  which  would  suggest  that  the  substance  is  an  anhydride  of 
bilirubin. 

Choletelin,  or  bilixanthin,  is  generally  regarded  as  the  final 
oxidation-product  of  the  common  bile-pigments.  It  is  an  amor- 
phous brown  substance,  which  is  soluble  in  alcohol,  ether,  chloro- 
form, and  in  solutions  of  the  alkaline  hydrates,  from  which  latter  it 
can  be  precipitated  l)y  the  addition  of  acids.  Its  formula  is  given 
as  Ci^HigXA- 


THE  BILE.  179 

Bilihumin  is  a  pigment  of  unknown  composition  that  has  been 
found  in  gall-stones.     It  is  insoluble  in  all  organic  solvents. 

Cholesterin. 

Cholesterin  is  not  exclusively  a  product  of  the  activity  of  the 
hepatic  cells,  but  is  found  in  other  tissues  as  well.  It  has  thus  been 
encountered  in  tiie  red  corpuscles  of  the  blood,  in  the  plasma,  in  the 
yolk  of  eggs,  in  the  semen,  and  in  the  secretion  of  the  sebaceous 
glands,  and  is  especially  abundant  in  nerve-tissue.  In  the  vegetable 
world  also  cholesterin  is  distributed.  The  liver  is  probably  the 
organ  through  which  the  substance,  wherever  formed,  is  eliminated. 
Ultimately  it  appears  in  the  feces.  In  the  urine  it  is  found  only 
under  exceptional  conditions,  and  then  only  in  very  small  amounts. 
Of  its  mode  of  formation  nothing  is  known,  but  it  is  interesting  to 
note  that  wherever  cholesterin  is  found  lecithin  is  likewise  observed. 
In  the  brain  a  considerable  amount  of  the  substance  occurs  in  com- 
bination with  a  fatty  acid,  cholic  acid,  from  which  it  can  only  be 
separated  by  saponification. 

The  amount  of  cholesterin  which  is  found  in  the  bile  represents 
about  2  per  cent,  of  the  total  solids.  Normally  it  is  held  in  solution 
owing  to  the  presence  of  the  biliary  acids,  but  under  pathologic  con- 
ditions it  may  separate  out  in  crystalline  form,  either  in  the  gall- 
bladder itself  or  in  the  larger  hepatic  ducts,  and  then  gives  rise  to 
the  formation  of  stones.  Of  the  origin  of  these  concretions  we  know 
little.  Very  often  they  contain  a  nucleus  of  epithelial  cells  or  of 
bacteria,  around  which  the  cholesterin,  together  with  a  variable 
amount  of  bile-pigment  and  mineral  salts,  becomes  deposited.  It  is 
possible  that  they  result  owing  to  a  temporary  absence  of  the  biliary 
acids,  but  this  is  only  a  supposition.  The  stones  which  arc  usually 
found  in  man  are  for  the  most  part  very  rich  in  cholesterin,  Avhile 
the  pigment-stones  which  are  so  common  in  cattle  are  less  frequently 
seen  in  the  human  being. 

The  common  cholesterin  which  is  found  in  the  animal  body  has 
the  composition  C27II4-.OH  (Obermuller),  and  is  usually  regarded  as 
a  monatomic  alcohol.  Of  its  structure,  however,  nothing  is  known. 
It  combines  with  fatty  acids  to  form  compound  ethers  which  are 
analogous  to  the  fats,  and  in  this  form  also,  as  has  been  shown 
(page  67)  cholesterin  occurs  widely  distributed  in  the  animal 
world.  The  common  lanolin  of  wool-flit  thus  contains  large 
amounts  of  such  compound  ethers,  both  of  cholesterin  and  its 
isomeric  compound  isocholesterin.  On  treating  cholesterin  with 
concentrated  sulphuric  acid  the  substance  gives  rise  to  the  formation 
of  certain  hydrocarbons,  which  are  termed  clioIc^tcrUins,  and  which 
are  supposed  to  stand  in  a  close  relation  to  the  terpene  group.  With 
iodine  these  bodies  give  a  blue  color. 

Cholesterin  usually  occurs  in  the  form  of  colorless,  transparent 
plates,  with  ragged  margins  and  angles,  which  are  very  characteristic 


180  THE  DIGESTIVE  FLUIDS. 

It  is  practically  insoluble  in  water,  dilute  acids  and  alkalies,  but  dis- 
solves with  ease  in  ether,  chloroform,  benzol,  and  in  boiling  alcohol. 
From  its  ethereal  solutions  it  crystallizes  out  in  the  form  of  fine 
needles.  It  is  further  soluble  in  the  essential  and  fatty  oils,  as  also 
in  the  presence  of  biliary  acids.     Its  crystals  melt  at  145°  C. 

Tests. — Salkowski's  Test. — A  few  crystals  of  cholesterin  are 
dissolved  in  a  small  amount  of  chloroform  and  treated  with  an 
equal  volume  of  concentrated  sulphuric  acid.  The  solution  of 
cholesterin  then  first  assumes  a  blood-red  color,  and  then  gradually 
turns  to  a  violet  red,  while  the  sulphuric  acid  appears  dark  red  and 
shows  a  green  fluorescence.  On  pouring  the  chloroform  into  a 
shallow  porcelain  dish  it  turns  violet,  then  green,  and  finally  becomes 
yellow. 

The  Test  of  Liebermann-Burckhard. — If  a  small  amount 
of  cholesterin  is  dissolved  in  about  2  c.c.  of  chloroform  and  is 
treated  with  10  drops  of  acetic  acid  anhydride,  and  subsequently 
with  concentrated  sulphuric  acid,  the  solution  at  first  assumes  a  red, 
then  a  blue,  and  finally  a  green  color.  The  latter  develops  at 
once  if  cholesterin  is  present  only  in  traces. 

Isolation. — To  prepare  cholesterin  for  purposes  of  study,  it  is 
most  convenient  to  isolate  the  substance  from  cholesterin  stones. 
To  this  end,  the  concretions  are  finely  pulverized  and  extracted 
with  boiling  water  and  then  with  boiling  alcohol.  From  the 
alcoholic  extract  the  cholesterin  crystallizes  out  on  cooling,  and  is 
then  boiled  with  an  alcoholic  solution  of  sodium  hydroxid,  so  as  to 
saponify  the  fats  which  are  at  the  same  time  present.  The  alcohol 
is  then  distilled  off  and  the  residue  extracted  with  ether,  which 
removes  the  cholesterin  and  leaves  the  soaps  behind.  On  evaporate- 
ing  this  extract,  after  filtration,  the  substance  is  obtained  in  crystal- 
line fi)rm,  and  can  be  further  purified  by  recrystallization  from  a 
mixture  of  alcohol  and  ether. 

Other  Organic  Constituents  of  the  Bile. — In  addition  to  the 
bodies  already  described,  the  bile  contains  also  small  amounts  of 
lecithin,  of  palraitin,  stearin,  olein,  and  the  soaps  of  the  correspond- 
ing fatty  acids.  In  ox-bile  Lassar-Cohn  found  also  traces  of 
myristinic  acid,  C^^^O.^^,  which  has  heretofore  only  been  observed 
in  the  spermaceti  of  whales.  We  further  find  traces  of  urea,, 
and  occasionally  a  diastatic  ferment,  which  is  by  some  observers 
regarded  as  identical  with  ptyalin.  Its  presence,  however,  is  by 
no  means  constant,  and  it  can  scarcely  be  regarded  as  playing  a 
role  in  the  process  of  intestinal  digestion.  Larger  amounts  of  urea, 
according  to  Hammarsten,  are  found  in  the  bile  of  the  shark  and 
the  sturgeon. 

In  decomposing  bile  cholin,  glycerin-phosphoric  acid  and  tri- 
methvlamin  may  be  observed,  and  are  referable  to  the  decomposition, 
of  lecithin. 


THE  BILE.  181 


The  Biliary  Iron. 

If  we  recall  the  origin  of  bilirubin  from  hsematin,  we  should 
expect  to  find  in  the  bile  the  iron  which  is  liberated  during  the 
decomposition  of  the  latter.  Traces  of  iron,  indeed,  are  constantly 
present,  principally  in  combination  with  phosphoric  acid.  The 
amount,  however,  which  is  thus  eliminated  is  far  too  small  to  repre- 
sent that  which  must  of  necessity  be  set  free.  Kunkel  thus  found 
that  while  100  parts  of  hsematin  correspond  to  9  parts  of  iron,  only 
1.4-1.5  parts  of  iron  appear  in  the  urine  for  every  100  parts  of 
bilirubin.  But  even  if  we  add  to  this  the  amount  which  is  eliminated 
in  the  urine  and  that  which  is  excreted  through  the  intestinal  mucosa, 
we  still  tind  a  very  large  deficit,  and  we  are  accordingly  forced  to 
the  conclusion  that  the  greater  portion  of  the  iron  must  be  retained 
in  the  liver.  But  while  such  a  retention  must  of  necessity  occur,  we 
are  profoundly  ignorant  of  the  manner  in  which  it  is  accomplished. 
Of  the  form,  also,  in  which  the  iron  exists  in  the  liver  we  know  but 
little.  That  it  is  subsequently  utilized  in  the  construction  of  haemo- 
globin is  quite  likely,  but  not  proved.  Naunyn  and  Minkowski  have 
observed  that  following  poisoning  with  arseniuretted  hydrogen,  iron- 
containing  pigments  can  be  demonstrated  in  the  liver.  Latschen- 
berger  speaks  of  the  formation  of  (•holeglobins  as  antecedents  of  bili- 
rubin, and  of  the  simultaneous  appearance  of  iron-containing  melanins 
in  the  liver ;  and  Neumann  has  demonstrated  the  presence  of  an 
organic  iron  pigment,  h;iemosiderin,  in  old  extravasations  of  blood 
and  in  thrombi  together  with  hsematoidin  ;  but  of  the  true  nature 
of  all  these  substances  we  practically  know  nothing.  It  is  possible 
that  the  globin,  which  must  of  necessity  be  liberated  during  the 
decomposition  of  hsematin,  takes  up  the  iron  which  is  set  free  from 
the  latter ;  but  this  also  is  a  supposition,  and  further  researches  in 
this  direction  are  urgently  needed. 


CHAPTER   VIII. 

THE  PROCESSES  OF  DIGESTION  AND  RESORPTION. 

In  the  preceding  chapter  we  have  considered  the  various  digestive 
fluids  which  are  concerned  in  the  transformation  of  those  food- 
stuffs that  are  incapable  of  resorption  as  such  into  material  which 
the  body  can  utilize  for  purposes  of  nutrition,  and  we  have  seen  that 
the  most  important  agents  which  are  here  concerned  belong  to  the 
class  of  the  non-organized  ferments.  In  the  present  chapter  we 
shall  study  the  action  of  these  various  substances  upon  the  different 
classes  of  food-stuflFs  collectively  and  in  somewhat  greater  detail, 
and  shall  incidentally  also  consider  the  resorption  of  the  final  prod- 
ucts of  digestion  from  the  gastro-intestinal  canal. 

THE   DIGESTION   OF    THE    CARBOHYDRATES. 

The  digestion  of  the  carbohydrates  is  essentially  effected  in  the 
small  intestine  through  the  agency  of  the  amylolytic  ferment  of 
the  pancreas,  ptyalin,  and  the  inverting  ferments  maltase,  lactase, 
and  invertin,  which  are  in  part  also  furnished  by  the  pancreas,  but 
are  principally  found  in  the  enteric  juice.  In  those  animals  in 
which  ptyalin  occurs  in  the  saliva,  amylolysis  to  a  certain  degree 
also  takes  place  in  the  mouth  and  continues  in  the  stomach  until 
hydrochloric  acid  appears  in  the  free  state,  when  the  ferment  is 
rapidly  destroyed.  In  man,  however,  the  salivary  digestion  only 
plays  a  secondary  rule.  It  is  true  that  the  end-product  of  amylo- 
lytic digestion,  viz.,  maltose,  can  probably  always  be  demonstrated 
in  the  gastric  contents,  even  after  a  few  minutes  following  the 
ingestion  of  starch  ;  but  it  must  be  borne  in  mind  that  the  trans- 
formation of  starch  into  sugar  does  not  take  place  in  distinct 
phases,  but  that  one  molecule  of  the  substance  may  have  already 
been  changed  to  maltose,  while  another  is  as  yet  unaffected.  In 
determining  the  intensity  and  the  extent  of  amylolytic  activity  it 
is  hence  unwarrantable  to  draw  conclusions  from  mere  qualitative 
tests,  and  it  is  necessary  to  compare  the  amount  of  sugar  which  is 
actually  formed  with  the  amount  of  starch  that  has  been  ingested. 

In  the  majority  of  the  purely  carnivorous  animals,  as  has  been 
pointed  out,  the  saliva  contains- no  digestive  ferments,  and,  in  such, 
carbohydrate  digestion  takes  place  exclusively  in  the  small  intestine. 

Through  the  action  of  the  ptyalin  of  the  pancreatic  juice  or  of  the 
saliva,  as  the  case  may  be,  the  insoluble  starch  is  first  transformed 
into  soluble  starch  or  amidulin  (amylodextrin),  and  is  then  succes- 
182 


THE  DIGESTION  OF  THE  CARBOHYDRATES.  183 

sively  decomposed  by  hydrolysis  into  erythrodextrin,  achroodextrin, 
isomaltose,  and  finally  into  maltose,  as  shown  by  the  equations  : 

(1)  (Ci2H2oOio)54  +  3H,0  =  3[(Ci,H,oO,o)„.Cv2H220ii] 

Amidulin.  Erythrodextrin. 

(2)  3[(C,,H,oO,o)„.C,,H,,0„]  +  6H,0  =  9[(C,,H,oOio)5-C,,H,Ai] 

Jirythrodextrin.  Aehroodextrin. 

(3)  9[(C\,H,,oO,o)5.C,,H,Ai]  +  45H,0  -=  54Ci,H,,Oii  =  54C,,H,Ai 

Aehroodextrin.  Isomaltose.  Maltose. 

Glycogen  is  similarly  decomposed,  and,  like  starch,  gives  rise  to 
the  formation  of  isomaltose  and  maltose.  The  celluloses,  on  the 
other  hand,  are  not  aflFected  by  ptyalin,  nor,  indeed,  by  any  of  the 
digestive  fluids.  As  we  shall  see,  however,  they  undergo  a  certain 
kind  of  fermentation  under  the  influence  of  various  bacteria,  and 
as  a  result  we  find  that  in  herbivorous  animals,  at  least,  only  a  frac- 
tion of  the  ingested  cellulose  reappears  in  the  feces.  Thus  far  a 
transformation  into  maltose  or  glucose  has  not  been  observed  in  the 
intestinal  tract. 

It  is  stated  that  through  the  activity  of  the  bacteria  of  the  intesti- 
nal canal,  viz.,  their  ferments,  a  certain  amount  of  starch  is  trans- 
formed into  maltose,  and  that  inversion  of  disaccharides  to  mono- 
saccharides is  likewise  brought  about  in  this  manner.  To  what 
extent  this  bacterial  action  can  be  regarded  as  representing  an  actual 
digestion  is,  however,  an  open  question.  The  presence  of  bacteria 
in  the  intestinal  canal  is  certainly  not  imperative,  as  has  been  con- 
clusively established  by  Nuttall  and  Thierfelder ;  and  we  know, 
moreover,  that  bacterial  action  extends  far  beyond  the  action  of 
digestive  ferments,  and  that  the  further  changes  that  can  thus  be 
effected  do  not  serve  a  useful  purpose.  The  normal  inversion  of 
the  disaccharides  is  unquestionably  brought  about  by  the  invertin, 
maltase,  and  lactase,  which,  as  has  been  indicated,  are  partly  fur- 
nished hy  the  pancreas,  but  principally  by  the  enteric  juice.  This 
inversion  is  likewise  of  the  nature  of  a  hydrolytic  process,  and  may 
be  represented  by  the  general  equation  : 

C,Ji,,0„     +     H.,0     =:    2CeHiA 
Disaecliaride.  Monosaccharide. 

To  judge  from  certain  experiments  which  have  been  performed 
on  animals,  it  appears,  however,  that  amylolysis  at  least  can  also 
take  place  in  the  absence  of  the  principal  gland  by  which  the 
ptyalin  is  formed,  viz.,  the  pancreas.  For  we  find  that  following 
the  extirpation  of  this  organ  or  ligation  of  the  pancreatic  duct  dogs 
are  still  capable  of  utilizing  as  much  as  from  47  to  71  per  cent,  of 
the  starch  ingested.  As  the  dog's  saliva  contains  no  ptyalin,  the 
amylolysis  cannot  be  referable  to  a  converting  activity  of  the  salivary 
glands.  AYhether  in  such  cases  the  small  amount  of  the  ferment 
which  is  also  furnished  by  the  enteric  juice  is  sufficient  to  transform 
the  ingested  starch  into  maltose  is  questionable,  and  there  is  some 
reason  for  supposing  that  the  epithelial  cells  of  the   small  intestine 


184        THE  PROCESSES  OF  DIGESTION  AND  RESORPTION. 

are  likewise  capable  not  only  of  causing  the  transformation  of  disac- 
charides  into  monosaccharides,  but  also  of  inverting  dextrin  to 
maltose.  It  has  been  shown  that  in  aniinals  with  Thiry-Vella 
fistula  injected  solutions  of  starch  and  cane-sugar  rapidly  disappear, 
although  maltose  cannot  at  times  be  demonstrated  in  the  fluid.  In 
what  manner  this  change  is  effected  by  the  epithelial  cells  is  not 
known.  In  any  event,  however,  it  is  necessary  that  the  polysac- 
charides should  be  inverted  to  monosaccharides  before  passing  beyond 
the  mucous  membrane  of  the  gastro-intestinal  tract.  Resorption 
takes  place  primarily  through  the  specific  activity  of  the  epithelial 
lining  of  the  gastro-intestinal  mucosa.  The  monosaccharides  then 
enter  the  blood-current  and  are  carried  to  the  muscles  and  the  liver, 
where  they  are  transformed  into  glycogen  and  stored  in  a  manner 
analogous  to  the  reserve  starch  of  the  plant.  This  transformation, 
howev^er,  as  well  as  the  subsequent  fate  of  the  sugar,  we  shall  have 
occasion  to  study  in  greater  detail  in  a  subsequent  chapter. 

Neither  the  jjolysaccharides  nor  the  disaccharides  when  introduced 
into  the  blood-current  directly  can  be  utilized  by  the  body  as  such, 
and  they  are  accordingly  eliminated  in  the  urine  as  foreign  matter. 

The  extent  to  which  amylolysis  can  occur  in  the  intestinal  canal 
is  remarkable,  and  for  exceeds  the  ability  of  the  liver  and  the  muscle- 
tissue  to  transform  the  corresponding  amount  of  monosaccharides 
into  glycogen.  As  a  consequence,  the  percentage  of  circulating 
sugar  rises  beyond  the  normal  and  glucosuria  results.  That  disac- 
charides may  pass  the  intestinal  mucosa  without  being  inverted  is 
possible,  but  is  certainly  of  exceptional  occurrence.  In  such  cases 
we  must  imagine  that  the  intestinal  epithelium  has  lost  its  specific 
power  as  a  barrier  to  the  passage  of  the  sugars,  as  well  as  its  ability 
to  cause  their  inversion.  As  a  result  they  pass  this  barrier  by  diffii- 
sion,  and  probably  enter  both  the  blood-  and  the  lymph-current,  and 
are  then  eliminated  in  the  urine.  A  formation  of  glycogen  from  di- 
saccharides directly  is  apparently  not  possible. 

The  rapidity  with  which  resorption  takes  place  in  the  small  intes- 
tine seems  to  vary  with  the  character  of  the  sugar.  In  dogs  Aiber- 
tini  thus  found  that  of  100  grammes  of  glucose,  60  grammes  are 
absorbed  in  the  course  of  the  first  hour,  while  of  maltose  and  cane- 
sugar  from  70  to  80  grammes  and  of  lactose  only  :^0  to  40  grammes 
disappear  Avithin  the  same  period  of  time. 

The  ingestion  of  very  large  amounts  of  disaccharides  and  mono- 
saccharides leads  to  a  general  disturbance  of  intestinal  digestion  and 
results  in  diarrhoea.  A  corresponding  amount  of  starch,  on  the 
other  hand,  is  without  effect  in  this  respect.  This  is  no  doubt  owing 
to  the  fact  that  in  the  latter  case  inversion  and  resorption  proceed 
pari  passu,  so  that  the  bacteria  have  but  little  chance  of  setting  up 
fermentative  changes,  which  lead  to  the  formation  of  substances 
that  directly  increase  the  peristalsis  owing  to  their  irritating  prop- 
erties. In  the  presence  of  abnormally  large  amounts  of  sugars  as 
such,  on  the  other  hand,  resorption  is  not  sufficiently  rapid,  and  in 


DIGESTION   OF  THE  ALBUMINS.  185 

the  presence  of  the  increased  amount  of  pabulum  an  increase  of 
bacterial  fermentation  beyond  the  normal  takes  place.  As  a  result 
the  various  acid  decomposition-products  of  the  carbohydrates  such 
as  lactic  acid,  butyric  acid,  acetic  acid,  formic  acid,  succinic  acid, 
together  with  carbon  dioxide,  methane,  and  hydrogen,  are  formed  in 
increased  amounts,  and  are  responsible  for  the  resulting  pathological 
conditions. 

DIGESTION  OF  THE  ALBUMINS. 

The  digestion  of  the  albumins  takes  place  in  the  stomach  and 
in  the  small  intestines  under  the  influence  of  the  pepsin  and  the  hy- 
drochloric acid  of  the  gastric  juice,  and  the  trypsin  of  the  alkaline 
pancreatic  juice,  respectively.  We  know  that  the  presence  of  the 
former  is  not  altogether  necessary,  however,  and  that  the  })ancreatic 
juice  is  in  itself  quite  sufficient  to  accomplish  the  digestion  of  the 
albumins  alone,  but  under  normal  conditions  the  gastric  juice  also 
plays  a  part.  Of  the  relative  extent  to  which  the  one  or  the  other 
enters  into  this  process  our  knowledge  is  not  complete.  We  may 
imagine  that  in  the  stomach  a  primary  dissclution  of  the  solid  con- 
stituents of  the  food  takes  place,  and  that  the  soluble  products 
which  are  thus  formed  are  further  digested  by  the  pancreatic  juice. 
This  actually  occurs  to  a  certain  extent,  but  we  further  know  that 
in  the  stomach  certain  albuminous  food-stufls  are  decomposed,  with 
the  liberation  of  constituents  which  are  insoluble  in  the  gastric  juice, 
and  which  pass  the  pylorus  as  such  and  are  then  modified  by  the 
pancreatic  juice.  Tryptic  digestion,  moreover,  is  far  more  extensive 
than  peptic  digestion,  so  that  we  may  well  conclude  that  the  latter 
essentially  represents  a  preliminary  phase  of  digestion ;  and  that 
the  digestion  proper,  viz.,  the  transformation  of  the  albumins  into 
those  final  products  which  can  be  directly  utilized  by  the  body, 
occurs  under  the  influence  of  the  trypsin  of  the  pancreatic  juice. 

For  convenience'  sake,  we  shall  study  the  action  of  the  gastric 
juice  and  of  the  pancreatic  juice  separately  upon  the  various  classes 
of  albumins,  as  the  digestive  products  which  are  formed  are  somewhat 
different  in  the  different  classes.  In  every  case  we  shall  follow  the 
fate  of  these  various  substances  to  the  final  products,  as  we  obtain 
them  artificially  in  digestive  experiments  in  vitro ;  but  we  must 
bear  in  mind  that  such  experiments  cannot  reproduce  what  actually 
takes  place  in  the  living  body,  where  resorption  is  constantly  going 
on,  and  where  the  various  digestive  processes  in  a  manner  supple- 
ment each  other,  and  conditions  overlap.  Whenever  possible  we 
shall  attempt  to  point  out  where  gastric  digestion  probably  ceases  and 
pancreatic  digestion  begins,  but  such  an  attempt  must  of  necessity  be 
more  or  less  crude. 

Digestion  of  the   Native  Albumins. 

Gastric  Digestion. — In  the  stomach  the  native  albumins,  if  in- 
troduced in  the  coagulated  state,  are  first  transformed  into  a  soluble 


]86        THE  PROCESSES  OF  DIGESTION  AND  RESORPTION. 

form,  and  at  the  same  time  or  immediately  following  their  dissolu- 
tion they  undergo  the  process  of  denaturizatiou — /.  e.,  they  are  trans- 
formed into  syntonins  or  acid  albumins,  and  as  a  consequence  all 
individual  characteristics  which  previously  existed  are  lost.  This 
transformation  is  essentially  referable  to  the  hydrochhjric  acid  of  the 
gastric  juice,  andean  be  brought  about  artificially  in  the  absence  of 
pepsin.  In  such  an  event,  however,  a  higher  grade  of  acidity  and  a 
higher  temperature  are  required.  The  presence  of  the  pepsin  ob- 
viates such  a  necessity,  A  possible  explanation  of  this  phenomenon 
is  afforded  by  the  modern  doctrine  which  teaches  that  the  action  of 
enzymes  merely  consists  in  hastening  the  rapidity  of  reaction. 

On  continued  exposure  to  the  acid  gastric  juice  albumoses  appear 
and  finally  peptones. 

This  decomposition  may  be  compared  to  the  inversion  of  tiie  poly- 
saccharides to  monosaccharides,  and  here,  as  there,  intermediate 
products  are  formed  which  differ  nut  only  from  the  syntonins,  but 
also  from  each  other.  Kiihne  and  his  school,  who  have  largely 
contributed  to  our  knowledge  of  these  products,  have  suggested 
their  division*  into  two  classes,  viz.,  the  primary  and  secondary 
albumoses,  according  to  their  nearer  or  more  distant  relationship  to 
the  original  albumins.  He  recognized  two  primary  albumoses,  viz., 
proto-albumose  and  hetero-albumose,  each  of  which  on  further 
digestion  w'as  supposed  to  give  rise  to  a  deutero-albumose,  from 
which  in  turn  peptone  was  derived.  This  peptone,  which  resulted 
from  peptic  digestion,  was  regarded  as  a  unity  and  termed  ampho- 
pepfoiie.  In  it  the  hemi-  and  anti-complex  of  the  albuminous  mole- 
cule were  still  supposedly  united.  In  a  general  way  tiiis  concept  of 
the  cour-e  of  peptic  digestion  has  proved  correct,  but  it  has  been 
modified  in  several  important  particulars  within  recent  years.  Much 
of  our  present  knowledge  is  due  to  the  Strassburg  school,  notably  to 
Pick,  Zuntz,  Umber,  Alexander,  and  others.  Their  researches  have 
shown  that  quite  early  in  the  course  of  digestion  a  certain  proportion 
of  nitrogen  is  split  off  from  the  albuminous  material  in  the  form  of 
ammonia  or  of  a  compound  which  yields  ammonia  on  distillation 
with  magnesia.  At  first  this  is  small  in  amount ;  subsequently  it 
increases,  and  for  a  certain  time  it  then  remains  constant.  This 
nitrogen  represents  products  which  no  longer  give  the  biuret 
reaction,  and  which  undoubtedly  are  very  closely  related  to  the  end- 
products  of  digestion.  They  are  termed  pepfoids.  Coincidently 
the  primary  albumoses  appear,  of  which  we  now  recognize  three. 
These  are  proto-albumose,  hetero-albumose,'  and  a  third  which  Pick 
has  designated  as  gluco-albumose,^  from  the  fact  that  it  contains  the 
glucosamin  complex,  which  is  absent  in  the  two  first  mentioned  (in 
the  case  of  fibrin,  at  any  rate). 

'  The  term  first  usprl  for  this  was  clcntcro-all)umosft  R  or  albnmose  Ba.  Ilofmeister  sug- 
gests si/nalhumn.He  as  a  better  term,  since  there  is  a  possibility  that  albumins  may  exist  which 
may  yield  a  primary  albumose  of  this  order  containing  no  carbohydrate  group" (casein),  and 
that  on  the  other  hand  albumins  which  are  very  rich  in  carbohydrate  groups  may  jjossibly 
form  proto-  aud  hetero-albumoses  in  which  these  are  rei^resented. 


DIGESTION  OF  THE  ALBUMINS.  187 

On  further  digestion  the  primary  albumoses  give  rise  to  second- 
ary or  dcutero-albumose^,  of  which  several  forms  exist.  These  are 
designated  as  deutero-albumose  A  and  A',  deutero-albumose  B  and 
B',  and  deutero-albumose  C.  These  in  turn  give  rise  to  a  class  of 
bodies  which  in  part  give  the  biuret  reaction  and  in  part  not.  The 
former  may  be  collectively  termed  peptones,  while  the  latter  are 
spoken  of  as  peptoids.  These  peptoids  represent  the  antecedents  of 
binary  or  correspondingly  simple  complexes,  from  wiiicli  the  end- 
products  proper  result.  Examples  of  such  relatively  simple  bodies 
are  leucinimid,  (C,2H22N202) )  albamin,  a  dihexosamin  of  the  com- 
position C,2H2iN203 ;  and'arginin,  CyH^^N^02. 

Several  bodies  have  been  described  as  pejitones.  Pick  thus 
speaks  of  an  A  and  a  B  peptone  which  result  from  the  secondary 
albumoses  (with  the  exception  of  deutero-albumose  C),  and  which 
differ  from  each  other  in  their  behavior  toward  alcohol  and  iodo- 
potassic  iodide  in  saturated  ammonium  suljihate  solution,  peptone  A 
being  precipitated  by  both  reagents,  while  peptone  B  remains  in 
solution.  A  further  examination  of  the  bodies  in  question  has  not 
been  made. 

Siegfried  and  his  pupils  describe  two  pepsin  peptones  which  were 
obtained  from  fibrin.  They  are  designated  as  pepsin-fibrin  ])e]>tone 
a  and  ,9.  By  losing  water  the  ^9-peptone  passes  over  into  the  a- 
peptone : 

(C,iH,«N,Oio),  C,,n34Ne09 

O)  (<») 

On  further  decomposition  with  trypsin  the  a-peptone  then  yields 
tyrosin,  arginin,  and,  according  to  Siegfried,  also  two  other  peptones, 
which  he  terms  trypsin-fibrin  peptone  a  and  /9.  He  accordingly 
points  out  that  the  a-peptone  is  'a  true  ampho-peptone  in  the  sense 
of  Kiihne. 

Of  the  nature  of  the  peptoids  nothing  definite  is  known. 

The  end-products  of  ])eptic  digestion  are  qualitatively  the  same  as 
those  which  result  on  tryptic  digestion.  The  velocity  of  reaction  in 
the  case  of  pepsin  is,  however,  rhaterially  less  than  with  trypsin. 
On  long-continued  peptic  digestion  the  following  well-defined  prod- 
ucts have  been  ol)tained  :  leucin,  amidovalerianic  acid,  tyrosin, 
phenyl-alanin,  glutaminic  acid,  asparaginic  acid,  cystin,  lysin,  ska- 
tosin,  putrescin,  cadaverin,  oxy-phenyl-ethylamin. 

The  question  has  been  raised  whether  this  extensive  digestion  is 
really  due  to  pepsin  and  not  to  pseudopepsin.  This  question  must 
still  remain  open,  as  it  is  an  impossibility  as  yet  to  obtain  a  pepsin 
uncontaminated  by  a  pseudopepsin.  Ijangstein,  however,  remarks 
that  in  working  with  a  commercial  product  he  never  obtained  a 
tryptophan  reaction,  although  he  encountered  the  various  end-prod- 
ucts just  enumerated.  This  would  suggest  that  the  hydrolysis  was 
due  to  pepsin,  as  pseudopepsin  supposedly  leads  to  the  production 
of  tryptophan   among  its  products  of  decomposition. 


188        THE  PROCESSES  OF  DIGESTION  AND  RESORPTION. 

Anioiii!;  the  ])rodiicts  of  digestion,  finally,  there  must  be  men- 
tiont'd  the  so-called  anti-alhinnid  ot"  Kiiime.  This  still  bears  an 
albuminous  character  and  supposedly  contains  the  anti-group  of  the 
albuminous  molecule,  as  it  is  extremely  resistentto  the  further  action 
of  pepsin  hydrochloric  acid.  On  tryptic  digestion  it  is  thrown 
down  as  a  delicate  jelly-like  material — the  so-called  anti-albumid 
coagulum.     The  substance  does  not  give  Millon's  reaction. 

As  regards  the  extent  to  which  peptic  digestion  is  carried  in  the 
stomach  our  knowledge  is  not  complete.  Zunz  has  recently  studied 
this  subject  in  the  case  of  dogs  fed  on  meat.  From  these  observa- 
tions it  appears  that  the  coagulated  albumins  are  first  brought  into 
solution  ;  during  this  process  very  little  acid  albumin  is  formed,  but 
large  amounts  of  albumoses  appear,  besides  small  amounts  of  ]>ep- 
tones,  pe])toids,  and  possibly  also  crystalline  end-j)roducts.  The 
larger  portion  of  the  material  which  is  now  in  solution  then  passes 
over  into  the  small  intestine,  where  it  is  rapidly  further  decomposed 
and  absorbed.  A  smaller  fraction  is  absorbed  in  the  stomach 
directly,  and  here  the  more  distant  ])roducts  of  digestion  are  pri- 
marily concerned,  while  an  absorption  of  albumoses,  though  its  oc- 
currence cannot  be  denied,  is  nevertheless  much  more  difficult. 
Accordingly  we  find  in  the  liquid  contents  of  the  stomach  mostly 
albumoses  and  only  small  amounts  of  the  more  remote  digestive 
products.  Of  albumctses,  we  find  both  primary  and  secondary 
forms;  a  definite  relation  between  the  two  does  not  appear  to  exist. 
Peptones  are  either  present  in  traces  or  they  are  absent,  while  })ep- 
toids  are  constantly  encountered. 

As  regards  the  appearance  of  the  individual  digestive  products  in 
point  of  time  and  in  relation  to  each  other  the  following  facts  have 
been  ascertained  (the  observations  have  reference  to  crystallized  serum- 
albumin,  crystallized  egg-albumiil,  serum-globulin,  euglobulin, 
pseudoglobulin,  and  casein  (Zunzi.  The  solution  of  the  coagulated 
albumins  l)egins  at  once  after  exposure  to  pepsin  hydrochloric  acid 
at  the  temperature  of  the  body.  In  the  case  of  serum-albumin  and 
euglobulin  the  primary  albumoses  appear  after  nine  minutes,  together 
with  acid  albumin.  In  the  case  of  casein  albumoses  are  likewise 
found,  but  no  acid  albumin,  at  the  same  time.  Pseudoglobulin  and 
egg-albumin  aj)pear  to  be  more  resistant,  as  no  digestive  products 
can  be  demonstrated  so  early.  Twenty-six  minutes  from  the  begin- 
ning of  digestion  acid  albumin  and  proto-  and  hetero-albumose  can 
be  demonstrated  in  the  case  of  the  egg-albumin,  together  with  the 
deutero-albumose  B. 

Acid  all)umin  is  never  found  in  the  entire  absence  of  albumoses  ; 
and  it  is  interesting  to  note  that  primary  albumoses  may  be  present 
at  a  time  when  no  acid  albimiin  is  as  yet  demonstrable.  This  ob- 
servation may  be  explained  by  the  assumption  that  the  acid  albumin, 
as  soon  as  formed,  is  transformed  into  allnimoses.  But  op]wsed  to 
this  interpretation  is  the  fact  that  acid  albumin  is  only  transformed 
into  albumoses  after  a  comparatively  longtime,  and  does  not  give 


DIGESTION  OF  THE  ALBUMINS.  189 

rise  to  all  products  of  digestion  whicli  can  be  obtained  from  the 
primary  albumins  from  wliicli  the  acid  albumin  in  turn  is  derived. 
Zunz  was  thus  unable  to  isolate  the  deutero-albuniuses  A  in  all  ex- 
periments in  this  direction.  It  has  accordingly  been  suggested  that 
acid  albumin  does  not  represent  a  purely  denaturized  albumin,  but 
should  be  placed  on  the  same  level  with  the  primary  albumoses. 

Goldschmidt  maintains  the  vnew  that  the  formation  of  acid  albu- 
min occurs  witli  a  coincident  splitting  off  of  albumose  complexes. 
On  the  other  hand,  it  has  been  noted  that  under  certain  conditions 
the  formation  of  acid  albumin  can  be  prevented  even  though  diges- 
tion otherwise  proceeds  in  the  normal  manner,  and  that  then  the 
deutero-albumose  A  is  likewise  absent,  as  also  that  portion  of  the 
end-products  of  pej)tic  digestion  which  give  no  biuret  reaction. 
It  follows  that  the  formation  of  acid  albumin  as  an  intermediate 
product  in  the  formation  of  albumoses  is  not  essential,  even  though 
it  may  be  of  value  during  the  process  of  digestion  in  the  living 
organism. 

Tryptic  Digestion. — On  entering  the  small  intestine  the  acid 
gastric  contents  are  rendered  alkaline,  the  pepsin  is  destroyed,  and 
tryptic  digestion  begins. 

The  material  which  is  exposed  to  the  action  of  the  pancreatic 
juice  consists  in  part  of  the  ])rimary  albumoses  which  were  formed 
in  the  stomach,  in  part  of  Kiihne's  anti-albumid,  and  in  part  of 
syntonin  and  of  native  albumins,  in  solul)le  or  insoluble  form, 
which  have  escaped  the  action  of  the  gastric  juice.  The  latter  are 
first  dissolved,  and  together  with  the  syntonins  transformed  iuto 
alkaline  albuminate.  This  result,  analogous  to  tiie  formation  of 
the  syntonin  in  acid  solution,  as  well  as  the  further  decomposition 
of  the  alkaline  albuminate,  is  no  doubt  primarily  referable  to  the 
action  of  the  alkalies  of  the  pancreatic  juice,  and  merely  hastened 
by  the  ferment  which  is  at  the  time  present.  But  unlike  the  action 
of  the  gastric  juice,  tryptic  digestion  immediately  leads  to  the 
formation  of  deutero-albumoses  without  the  intermediary  produc- 
tion of  primary  albumoses  in  the  sense  of  Kiihne.  According  to 
older  views,  amphopeptone  is  then  formed,  from  which  hemi-  and 
anti-peptone  finally  result.  This  concept,  as  in  the  case  of  the 
gastric  digestion,  has  been  materially  modified  by  more  recent  in- 
vestigations. Followiufy  the  sta^e  of  deutero-albumoses,  here  as 
there,  products  are  formed  which  in  part  give  the  biuret  reaction 
and  in  part  not.  A  hemipe])tone  and  an  antipeptone  in  the  sense 
of  Kiihne  do  not  exist.  The  hemi-  groups  at  once  break  down  into 
amido-acids  and  tryptophan,  Kiihne's  antipeptone,  which  repre- 
sents that  portion  of  the  try])tic  digestive  products  which  cannot  be 
salted  out  by  ammonium  sulphate  either  in  neutral,  acid,  or  allcaline 
media,  but  which  can  be  precipitated  l)y  absolute  alcohol,  is  no 
chemical  unity.  Kutscher  has  demonstrated  in  the  case  of  fibrin 
that  the  antipeptone  consists  to  the  extent  of  30  per  cent,  of  arginin, 
Ivsin,  and  histidin.     In  addition  he  found  small  amounts  of  leucin, 


190        THE  PROCESSES  OF  DIGESTION  AND  RESORPTION. 

tyrosin,  asparaginic  acid,  and  still  other  bodies  which  were  not 
identified. 

Peptones,  analogous  to  those  which  Siegfried  has  described  in 
the  case  of  peptic  digestion,  are  here  also  formed.  These  peptones, 
according  to  Siegfried,  do  not  contain  the  tyrosin  group  and  ener- 
getically resist  the  further  action  of  trypsin.  In  this  sense  they 
correspond  to  Kiihne's  concept  of  antipeptone.  From  fibrin  he 
obtained  two  bodies  of  this  order,  which  he  terms  a-  and  /9-trvpsin- 
tibrin  peptone,  Ci^Hj^NgOj  and  CnHigNgOj.  They  are  both  active 
acids ;  they  color  blue  litmus  paper  red  and  decomj)ose  carbonates 
with  the  formation  of  COg.  On  decomposition  with  acids  they 
yield  among  other  products  arginin,  lysin,  glutaminic  acid,  and 
possibly  also  serin. 

From  glutin  Siegfried  obtained  a  glutin-trypsin-peptone, 
Cj9H3oNg09.  On  hydrolysis  he  obtained  from  this  a  basic  pejitone 
which  he  terms  ghdohyrin  (to  xuf/o-,  the  nucleus),  and  wiiich  on 
further  cleavage  yields  arginin,  lysin,  glutaminic  acid,  and  probably 
glycocoll.  He  supposes  that  the  kyrin  molecule  consists  of  one 
molecule  of  arginin,  one  molecule  of  lysin,  one  of  glutaminic  acid, 
and  two  of  glycocoll. 

In  this  connection  it  is  interesting  to  note  that  on  tryptic  digestion 
of  various  albumins  E.  Fischer  and  Abderhalden  obtained  a  poly- 
peptide which  "was  resistant  to  the  further  decomposition  by  means 
of  trypsin,  but  which  yielded  a  large  amount  of  «-pyrrolidin  car- 
bonic acid  and  phenyl-alanin  ;  on  hydrolysis  with  boiling  hydro- 
chloric acid,  this  polypeptide  no  longer  gives  the  biuret  reaction. 
This  reaction  disappears  altogether  on  long-continued  tryptic 
digestion,  from  which  we  can  conclude  that  Siegfried's  peptones 
also  must  have  undergone  further  cleavage. 

The  end-products  of  tryptic  digestion  are,  as  already  pointed  out, 
qualitatively  the  same  as  those  which  result  on  peptic  digestion, 
with  the  exception  of  tryjitophan — skatol-amido-acetic  acid,  which 
may  in  a  measure  be  regarded  as  characteristic  of  tryptic  action. 
The  essential  difference  is  a  matter  of  velocity  of  reaction  ;  this  is 
materially  greater  with  trypsin  than  Avith  pepsin. 

The  chyme  from  the  stomach  reaches  the  duodenum  at  the 
beginning  of  the  fourth  hour  after  a  full  meal  ;  the  height  of  pan- 
creatic digestion  lies  between  the  third  and  the  fifth  hour. 

In  the  account  of  the  process  of  albuminous  digestion,  as  out- 
lined in  the  foregoing  pages,  it  has  in  a  manner  been  assumed  that 
the  intermediary  products  of  digestion  are  the  same,  irrespective 
of  their  origin.  Strictly  speaking,  this  is  probably  not  correct, 
although  in  the  absence  of  quantitative  studies  of  their  decomposi- 
tion-products it  is  scarcely  warrantai)le  to  make  ])ositive  statements. 
Their  identity,  however,  is  rendered  unlikely  by  the  varying 
degree  to  which  the  various  primary  radicles  are  represented  in  the 
different  native  bodies. 


DIGESTION  OF  THE  ALBUMINS.  191 


Digestion  of  the  Proteids. 

The  digestion  of  tlie  proteids,  or  of  the  nucleo-alhumins,  the 
glucoproteids,  and  tlie  hEemoglobins  at  least,  like  that  of  the  native 
albumins,  begins  in  the  stomach.  Here  the  separation  of  the  non- 
albuminous.  i)airling  is  first  effected,  and  is  then  followed  by  the 
digestion  of  the  liberated  albumins.  This  digestion  is  in  all  respects 
analogous  to  that  of  the  native  albumins  proper.  Syntonins  are  first 
formed,  then  primary  albumoses,  subsequently  secondary  albumoses, 
and  finally  peptones — /.  e.,  bodies  which  still  give  the  biuret  reac- 
tion, but  which  in  contradistinction  to  the  albumoses  are  not  pre- 
cipitated by  salting  with  ammonium  sulphate.  The  individual 
products  which  thus  result  from  the  proteids  have  not  as  yet  been 
studied  with  the  same  care  as  those  which  are  derived  from  the 
native  albumins,  but  it  is  likely  that  here  also  Kiihne's  schema  of 
digestion  does  not  apply  in  its  original  form.  Individual  differ- 
ences also  no  doubt  exist  between  the  various  digestive  products 
according  to  their  origin,  but  of  these  also  we  know  but  little. 

Of  special  interest  are  the  earlier  phases  of  digestion  of  the 
casein  of  milk.  This  normally  exists  in  the  milk  in  solution  as  a 
neutral  calcium  salt.  In  the  stomach  a  transformation  into  the 
corresponding  acid  salt  is  then  first  effected  by  the  hydrochloric 
acid  of  the  gastric  juice,  and  followed  by  the  action  of  the  chymosin. 
According  to  Hammarsten,  this  effects  a  partial  decomposition  of  the 
soluble  acid  salt  with  the  formation  of  calcium-paracasein,  and  a 
small  amount  of  an  albumose-like  posset  albumin.  The  paracasein 
is  then  precipitated  and  decomposed,  with  the  formation  of  the  cor- 
responding paranuclein  and  the  allmminous  pairling. 

Of  the  fate  of  the  non-albuminous  components  of  the  proteids 
but  little  is  known.  The  paranuclein  of  casein,  it  is  stated,  undergoes 
solution  on  continued  digestion  in  vitro,  but  is  at  the  same  time  de- 
composed with  the  formation  of  a  small  amount  of  orthophosphoric 
acid  and  an  organic  acid,  which  likewise  contains  phosphorus.  Of 
this,  however,  nothing  further  is  known  (see  also  page  57). 

The  nucleins  proper  are  not  digested  in  the  stomach  and  remain 
undissolved. 

Under  the  influence  of  the  pancreatic  juice  casein  is  digested  in 
very  much  the  same  manner  as  with  the  gastric  juice,  but  in  this 
case  the  transformation  into  paracasein  is  brought  about  through 
the  influence  of  the  chymosin  of  tlie  pancreas  in  an  alkaline  medium. 
Caseoses  then  result  as  with  the  common  native  albumins,  and  finally 
peptones  are  formed. 

The  glucoproteids  and  the  haemoglobins  are  decomposed  as  in  the 
case  of  the  gastric  juice,  and  the  albuminous  components  further 
digested  like  tlie  native  albumins.  The  individual  products,  how- 
ever, which  are  thus  formed  have  not  as  yet  been  studied  in  detail. 

The  true  nucleins,  which,  as  we  have  seen,  escape  gastric  diges- 
tion and  which  do  not  undergo  solution  in  the  stomach,  are  dissolved 


192         THE  PROCESSES   OF  DIGESTION  AND   RESORPTION. 

by  the  pancreatic  juice  and  are  decompoped  with  the  liberation  of 
the  contained  nuchnnic  acids  and  the  albuminous  radicles.  The 
latter  are  further  diijjested  in  the  usual  manner.  The  paranucleins 
similarly  undergo  dissolution,  and  are  probably  decomposed  as 
already  indicated.  Of  the  subsequent  fate  of  the  non-albuminous 
pairliiigs  of  the  proteids  in  general,  however,  but  little  is  known. 

Digestion   of  the   Albuminoids. 

The  only  all)uminoids  wdiich  are  digested  in  the  stomach  in  the 
case  of  the  higher  vertebrate  animals  are  collagen  and  elastin. 
Both  then  give  rise  to  protogelatose  and  proto-elastose,  respectively, 
while  curiously  enough  hetero-albumoses  are  not  formed.  The  cor- 
responding deutero-albumoses  then  result.  But  while  the  deutero- 
gelatose  subsequently  gives  rise  to  the  formation  of  peptone — the 
so-called  glutin-peptone — a  similar  transformation  of  the  deutero- 
elastose  apparently  does  not  occur. 

In  the  small  intestine,  under  the  influence  of  the  pancreatic 
juice,  collagen  and  elastin  can  also  be  digested,  and  it  is  note- 
worthy that  the  transformation  of  the  gelatins  into  glutin-peptone 
is  apparently  more  readily  effected  than  that  of  any  other  albumin- 
ous substance.  Unlike  the  gastric  juice,  however,  the  pancreatic 
secretion  is  in  itself  not  capable  of  transforming  the  native  collagen 
into  gelatin.  This  change  must  hence  be  first  effected  artificially 
or  in  the  stomach  before  its  further  digestion  can  occur.  The 
peptonization  of  elastin  in  the  pancreatic  juice  likewise  ceases  with 
the  formation  of  deutero-elastose,  while  gelatin  is  transformed  in 
vitro,  at  least,  into  glutin-peptone.  Accorduig  to  Kiihne  and  Chit- 
tenden, this  is  not  further  decomposed  by  the  trypsin.  This  is 
rather  remarkable,  as  on  hydrolytic  decomposition  with  mineral 
acids  gelatin  yields  leucin,  asparaginic  acid,  glutaminic  acid,  and 
considerable  amounts  of  glycocoll.  Reich-Herzberger,  however, 
has  recently  announced  that  a  slight  formation  of  leucin  takes 
place  nevertheless  during  the  action  of  trypsin  on  gelatin.  The 
existence  of  aromatic  groups  in  the  original  molecule,  on  the  other 
hand,  is  very  doubtful,  and  as  a  matter  of  fact  it  is  imjwssible  to 
obtain  either  tyrosin,  or  indol,  or  skatol,  from  the  substance,  even 
on  bacterial  decomposition.  Hexon  groups,  however,  are  largely 
present. 

The  fact  that  neither  elastin  nor  collagen  (gelatin)  gives  rise  to 
the  formation  of  hetero-albumoses  is  of  special  interest  in  view  of 
the  fact  that  both  substances  cannot  be  regarded  as  true  food-stuffs, 
viz.,  they  are  unable  to  maintain  the  nitrogenous  equilibrium  of  the 
higher  animals  when  exclusively  used.  Whether  or  not  this  is  due 
to  the  absence  of  the  aromatic  group  in  the  gelatin,  and  its  presence 
only  in  small  amounts  in  elastin,  is  not  decided.  The  hexon  bases 
are  manifestly  of  no  moment  in  this  connection,  as  gelatin,  at  least, 
yields  rather  more  arginin  than  any  of  the  common  albuminous  food- 


RESORPTION  OF  PRODUCTS  OF  DIGESTION.  193 

stuffs,  and,  as  I  have  pointed  out,  Ellinger  has  shown  that  the 
hexon  bases  alone  are  likewise  not  capable  of  maintaining  nitro- 
genous equilibrium.  Future  researches,  no  doubt,  will  explain  this 
essential  difference  between  the  albumins  proper,  including  the 
proteids  and  the  albuminoids. 

A  digestion  of  other  albuminoids,  notably  of  the  keratins,  does 
not  take  place  in  the  small  intestine  of  the  higher  animals,  while 
some  of  the  invertebrates,  such  as  the  common  house  moth,  are 
manifestly  capable  of  utilizing  these  also  for  purposes  of  nutrition. 
In  contradistinction  to  collagen  and  elastin,  the  keratins  yield  a 
relatively  large  amount  of  tyrosin,  in  addition  to  leucin,  aspara- 
ginic acid,  and  glutaminic  acid,  on  hydrolytic  decomposition. 

RESORPTION  OF    THE   PRODUCTS  OF   PROTEOLYTIC 

DIGESTION. 

The  resorption  of  the  products  of  proteolytic  digestion  is  essentially 
the  function  of  the  intestinal  mucosa.  The  gastric  mucous  mem- 
brane probably  plays  a  secondary  role  only,  and,  as  I  have  already 
pointed  out,  normal  nutrition  can  be  maintained  in  the  absence  of 
the  stomach.  Of  the  manner  in  which  resorption  takes  place  we 
know  very  little.  That  the  process  is  not  one  of  simple  diffusion 
seems  to  be  definitely  established,  and  there  is  good  evidence  to 
show  that  the  epithelial  cells  lining  the  mucous  membrane  are 
actively  concerned  in  the  event. 

In  the  past  much  uncertainty  existed  in  reference  to  the  form 
in  which  the  albumins  were  absorbed,  and  it  was  generally  taught 
that  digestion  proceeded  as  far  as  the  formation  of  albumoses  and 
"  peptones,"  in  the  older  sense  of  the  term,  and  that  the  reconstruc- 
tion of  the  albuminous  molecule  then  occurred  in  the  gastro-in- 
testinal  mucous  membrane.  This  conclusion  appeared  to  be  well 
suiiported  by  the  observation  of  Hofmeister,  Neumeister,  and 
Salvioli,  that  "peptones"  will  disappear  from  a  solution  in  the 
presence  of  particles  of  intestinal  mucous  membrane.  More  recent 
experiments  by  Cohnheim  have  thrown  a  new  light  on  this  observa- 
tion and  have  materially  contributed  to  our  understanding  of  resorp- 
tive  processes.  His  researches  centre  in  the  discovery  of  a  new 
ferment,  erepsin,  in  the  i-ntestinal  mucosa,  which  is  capable  of 
hydrolyzing  acid  albumin,  albumoses,  and  peptones,  but  which  is 
without  affect  upon  the  native  albumins. 

Erepsin. — To  isolate  the  ferment,  the  intestinal  mucosa  of 
recently  killed  animals  is  scraped  off  with  a  piece  of  glass,  triturated 
with  sand,  extracted  repeatedly  from  one-half  to  twelve  hours  with 
alkaline  normal  salt  solution,  and  finally  pressed  out  in  a  tincture 
press.  Pressings  and  extract  are  united  and  treated  with  a  saturated 
solution  of  ammonium  sulphate,  in  the  proportion  of  three  volumes 
of  the  salt  solution  to  two  of  the  extract.  A  heavy  precipitate 
results  on  standing,  which  is  filtered  off,  suspended  in  water,  and 

13 


194  RESORPTION  OF  PRODUCTS  OF  DIGESTION. 

dialyzed.  The  greater  portion  of  the  albumins  remains  undissolved, 
■while  a  smaller  amount  together  Avith  the  ferment  passes  into 
solution.  The  residue  is  again  extracted,  the  extracts  are  united,  and 
dialyzed  free  from  the  sulpliate  (three  to  four  days).  This  solution 
is  quite  active  and  rapidly  changes  albumoses  and  peptones  into 
material  which  no  longer  gives  the  biuret  reaction  and  is  in  great 
part  precipitated  by  phosphotungstic  acid  in  crystalline  form. 

The  ferment  is  destroyed  by  boiling,  and  is  much  impaired  in 
its  activity  by  exposure  to  a  temperature  of  G3°  C.  for  two  hours. 
It  decomposes  peptone  in  feebly  alkaline  or  neutral  media,  -while 
it  is  inactive  in  an  acid  medium.  Acetic  acid  does  not  destroy  the 
ferment  within  one  hour.  Prolonged  contact  with  hydrochloric 
acid  (of  course,  always  in  dilute  solution)  seems  to  destroy  it. 
Alcohol  impairs  its  activity  very  materially. 

Among  the  products  of  decomposition  resulting  from  the  action 
of  erepsin  on  syntonin  C(jhnheim  obtained  arginin,  lysin,  histidin, 
leucin,  tvrosin,  and  ammonia.  Especially  important  is  the  fact 
that  the  amount  of  ammonia  which  is  obtained  on  distillation  with 
magnesia  from  the  mixture  of  decomposition-products  is  the  same 
as  is  obtained  on  decomposition  with  acids,  as  also  with  try])sin, 
showing  that  the  decomposition  of  the  nitrogenous  molecule  does 
not  extend  beyond  the  stage  of  amido-acids. 

As  regards  the  action  of  erepsin  on  the  various  albumoses  and 
peptones,  Cohnheim  ascertained  that  they  are  all  decomposed  by 
the  erepsin  to  the  point  where  the  biuret  reaction  is  no  longer 
obtained  ;  though  with  varying  rapidity.  The  native  albumins  are 
in  no  ways  affected,  but,  very  curiously,  casein  is  decomposed. 
This  is  noteworthy  in  view  of  Gmelin's  observation  that  suckling 
dogs  secrete  no  pepsin. 

The  protamins  are  decomposed  entirely  like  the  albumoses  and 
peptones,  while  the  histons  are  only  aifected  in  part,  which  coin- 
cides with  the  position  which  the  histons  occupy  midway  between 
the  protamins  and  the  true  albumins. 

While  Cohnheim  assumes  that  the  activity  of  erepsin  is  mainly 
displaved  within  the  cells  of  the  intestinal  mucosa,  there  is  some 
evidence  to  show  that  a  portion  of  the  ferment  is  secreted  into  the 
lumen  of  the  gut  and  may  thus  become  active  outside  of  the  cells 
also  (Salaskin). 

To  demonstrate  the  action  of  erepsin,  it  is  only  necessary  to  add 
from  15  to  20  c.c.  of  an  extract  of  the  intestinal  mucosa,  prepared 
as  described  above,  to  a  small  amount  of  a  solution  of  pe])tone 
(Witte),  and  to  test  every  ten  to  fifteen  minutes  for  the  biuret  reaction. 
As  this  disap])ears,  it  may  be  shown  that  the  amount  of  material 
which  vields  a  crystalline  precipitate  with  phosphotungstic  acid 
irraduallv  increases,  while  the  gelatinous  preci])itate,  referable  to 
the  albumoses,  diminishes  in  amount  and  ultimately  disappears. 
Anv  coagulable  albumins  that  may  be  present  should  be  removed 
before  testing. 


RESORPTION  OF  PRODUCTS  OF  DIGESTION.  195 

Cohnheini's  experiments  thus  place  the  observation  that  peptone 
disappears  from  its  sohitions  in  tlie  presence  of  pieces  of  intestinal 
mucous  membrane,  in  a  new  ligiit.  For  it  disapj)ears,  not  because 
the  epithelial  cells  reconstruct  albumins  from  this  source,  but  be- 
cause the  erepsin  causes  its  further  cleavage  to  products  which  no 
longer  give  the  biuret  reaction.  Where  and  how  the  reconstruction 
of  the  albuminous  molecule  then  takes  place  is  not  known  ;  but 
there  can  be  no  reasonable  doubt  that  this  occurs  from  the  end- 
products  of  proteolytic  digestion,  and  that  albuminous  cleavage 
and  reconstruction  thus  go  on  in  a  manner  perfectly  analogous  to 
what  we  have  already  seen  in  the  case  of  the  carbohydrates. 

Of  great  interest  in  this  connection  are  certain  experiments  of 
Lowi,  in  which  he  succeeded  in  maintaining  the  nitrogenous  equi- 
librium of  a  dog  by  feeding  with  a  mixture  of  the  crystalline 
decomposition-])roducts,  the  result  of  pancreatic  autolysis,  in  which 
the  biuret  reaction  could  no  longer  be  demonstrated.  Henderson 
and  Dean  performed  a  similar  experiment  with  a  similar  result, 
although  they  merely  conclude  that  they  have  established  a  marked 
protein-sparing  action  on  the  part  of  the  cleavage-products.  The 
hexon  bases  alone,  as  Ellinger  has  shown,  are  not  capable  of  main- 
taining nitrogenous  equilibrium. 

The  question  whether  or  not  native  albumins  can  be  absoi'bed  as 
such  by  the  gastro-intestinal  mucosa  can  be  answered  in  the  affirma- 
tive. Ascoli  and  Vigano  have  thus  shown  by  the  aid  of  the 
precipitin  reaction  that  native  as  well  as  denaturized  albumins  can 
pass  the  gastro-intestinal  mucosa  and  enter  the  lym])h  and  blood, 
while  retaining  at  least  a  portion  of  their  biological  characteristics. 
To  what  extent  this  passage  of  native  albumins  occurs  under  normal 
conditions  remains  to  be  seen. 

Whether  or  not  albumoses  also  can  pass  the  intestinal  mucosa 
without  undergoing  further  cleavage  has  not  been  satisfactorily  ascer- 
tained. Embden  and  Knoop  maintain  that  this  is  possible,  and 
Langstein  has  shown  that  one  or  more  albumoses  can  actually  be 
demonstrated  in  the  blood.  Further  investigations  in  this  direction, 
however,  are  necessary  before  any  definite  conclusions  can  be 
reached. 

The  observation  that  albumins  when  introduced  into  the  blood- 
current  directly  can  be  utilized  to  a  very  great  extent  cannot  be 
surprising  in  the  case  of  those  albumins  which  are  normally  found 
here.  But  Oppenheimer  has  shown  that  this  also  occurs  to  a  certain 
extent  in  the  case  of  albumins  which  are  foreign  to  the  animal  body, 
such  as  egg-albumin.  When  introduced  beyond  a  certain  amount, 
however,  such  albumins  are  eliminated  in  the  urine  ;  but  very  curi- 
ously the  power  of  retaining  egg-albumin,  for  example,  can  be  greatly 
increased  by  frequently  repeated  hypodermic  or  intravenous  injec- 
tions. 

In  connection  with  the  question  regarding  the  reconstruction  of 
the  albuminous   molecule,   certain   observations   made  by   Russian 


196  RESORPTION  OF  PRODUCTS  OF  DIGESTION. 

writers  especially  deserve  consideration.  Danilewsky  has  thus 
shown  that  it  is  possible  by  means  of  chymosin  to  produce  preci})i- 
tates  in  concentrated  solutions  of  albumoses,  and  Okunew  could 
demonstrate  that  this  action  is  referable  to  the  cliymosin  itself  and 
not  to  any  other  contaminating  ferment.  It  was  further  shown 
that  both  proto-  and  hetero-g,lbumose  are  affected  by  the  chymosin 
and  not  the  deutero-albumoses.  The  resulting  products  Sawjalow 
has  termed  plaste'uis,  and  he  regards  these  as  allumiins  sui  generis. 
Later  Kurajeif  then  showed  that  papayotin  has  a  similar  action, 
but  that  tiie  secondary  albumoses  represent  the  most  appropriate 
material  from  which  papayotin-plastei'n,  can  be  prepared.  On 
peptic  or  papayotin  digestion  this  product  again  yields  secondary 
albumoses. 

Preceding  Cohnheim's  investigations,  these  experiments  were 
interpreted  as  indicating  the  manner  in  which  the  reconstruction  of 
the  albuminous  molecule  might  possibly  occur  in  the  intestinal 
mucosa.  In  the  light  of  Cohnheim's  discovery  of  erepsin,  however, 
this  construction  falls  away.  They  are  of  interest,  however,  in 
showing  that  })rotein  syntheses  also  can  be  effected  by  ferments,  for 
as  such  we  must  undoubtedly  regard  the  formation  of  the  plastei'ns. 

THE  DIGESTION  OF  FATS. 

The  important  role  of  the  pancreas  in  the  digestion  of  fats  is 
apparent  from  the  experiments  of  Minkowski  and  Abelmann,  who 
could  show  that  following  extirpation  of  the  pancreas  in  dogs  the 
absorption  of  fats  ceases  altogether,  with  the  exception  of  butter- 
fat,  of  which  from  28  to  5'')  per  cent,  can  still  be  utilized.  Other 
observers  have  obtained  results  which  differ  somewhat  from  those 
of  Minkowski ;  but  in  all  investigations  it  could  at  least  be  demon- 
strated that  in  the  absence  of  the  pancreatic  juice  the  absorption  of 
fats  is  impeded.  This  is  the  result  of  the  elimination  of  lipase 
(steapsin).  Under  normal  conditions  the  lipase  of  the  pancreatic 
jui(!e  causes  the  cleavage  of  fats  into  glycerin  and  fatty  acids,  which 
are  then  absorbed  and  reconstructed  into  neutral  fats  by  the  intra- 
cellular lipase,  in  consequence  of  the  reversible  action  of  which  this 
is  capable  (Kastle  and  Loevenhart). 

The  digestion  of  fats  is  facilitated  by  the  presence  of  bile.  This 
is  ap))arent  from  experiments  in  which  the  bile  was  prevented  from 
entering  the  intestinal  tract,  when  it  could  be  demonstrated  that 
only  the  seventh  part  to  one-half  of  the  fat  was  resorbed,  while  the 
remainder,  principally  in  the  form  of  fatty  acids,  appeared  in  the 
feces.  The  former  view,  according  to  wdiich  the  bile  fatalitates  the 
emulsification  of  the  fats  and  rendfTs  the  intestinal  wall  more  per- 
meable, probably  does  not  exjjress  the  actual  function  of  the  bile  in 
this  respect.  Moore,  Rockwood,  and  notaljly  PHiiger  have  shown 
that  its  principal  import  in  this  direction  is  referable  to  the  readi- 
ness with  which  it  dissolves  soaps,  and  through  their  aid  also  free 


AUTOLYSIS.  197 

fatty  acids,  especially  oleic  acid,  Avhich  in  turn  dissolves  stearic  acid 
and  palmitic  acid. 

The  lecithins,  like  the  fats,  are  decomposed  by  steapsin  into  their 
components,  viz.,  into  glycerin-phosphoric  acid,  the  corresponding 
fatty  acids,  and  cholin.  The  former  is  then  absorbed,  and  appears 
in  part  at  least  in  the  urine  as  such.  The  fatty  acids  after  saponifi- 
cation are  similarly  absorbed  and  reconstructed  into  neutral  fats, 
while  cholin  is  decomposed  by  the  bacteria  which  are  present  in 
the  intestines,  with  the  formation  of  carbon  dioxide,  methane,  and 
ammonia. 

AUTOLYSIS. 

By  autolysis  is  meant  the  self-digestion  of  tissues  in  the  absence 
of  micro-organisms.  The  phenomenon  is  referable  to  the  action  of 
intracellular  ferments,  and  is  of  universal  occurrence  both  in  the 
animal  and  the  vegetable  world.  The  changes  which  occur  are 
essentially  postmortem  changes ;  but  it  is  possible  that  sometliing 
similar  occurs  during  life. 

As  I  have  already  pointed  out,  the  ferments  in  question  are  more 
or  less  specific  in  their  action,  and  albumins  which  are  foreign  to  a 
certain  cell  are,  generally  speaking,  attacked  with  greater  difficulty 
by  the  autolytic  ferments  of  that  cell  than  the  albumins  of  homol- 
ogous cells.  The  proteolytic  ferments  of  hepatic  tissue  thus  attack 
the  albumins  of  lung-tissue  only  very  slowly. 

The  proteolysis  proceeds  as  in  the  case  of  the  digestive  ferments 
and  the  end-products  are  the  same.  In  the  case  of  the  pancreas 
the  following  products  have  been  obtained  :  ammoniii,  leucin,  tyro- 
sin,  as])araginic  acid,  glutaminic  acid,  arginin,  lysin,  histidin,  cadav- 
erin,  oxy-phenyl-ethylamin,  skatosin,  uracil,  guanin,  adenin,  xanthin, 
hypoxanthin,  cliolin,  etc.  Especially  interesting  is  the  formation 
of  oxy-phenyl-ethylamin,  which  is  derived  from  tyrosin  through 
loss  of  COo,  as  it"  demonstrates  the  possibility  of  a  fermentative 
splitting  off  of  CO2  in  the  body  without  the  simultaneous  interven- 
tion of  oxygen  and  water. 

The  skatosin  which  Baum  isolated  from  the  products  of  pancreatic 
autolysis  has  the  composition  CinHigN2C^2-  I^  contains  two  amido 
and  two  hydroxyl  groups,  and  is  probably  identical  with  a  body 
which  Langstein  found  among  the  final  products  of  peptic  digestion 
in  the  case  of  serum-albumin. 

The  occurrence  of  xanthin  bases  among  the  products  of  autolysis 
shows  that  a  nuclease  also  may  have  been  active. 

Autolysis  of  lymph-glands  gives  rise  to  ammonia,  leucin,  tyrosin, 
thymin,  and  uracil.     Purin  bases  were  here  not  obtained  (Reh). 

That  tryptophan  can  also  be  formed  during  the  autolysis  of  the 
gastric  mucous  membrane  has  been  shown  bv  Gliissner ;  during  the 
process  both  pepsin  and  chymosin  are  destroyed. 


CHAPTER    IX. 

ANALYSIS  OF  THE  PRODUCTS  OF  ALBUMINOUS  DIGESTION. 

To  demonstrate  the  formation  of  the  various  products  of  albu- 
minous digestion  and  to  separate  the  individual  substances  from  each 
other,  the  following  plan  of  work  may  be  adopted  : 

THE  PRODUCTS  OF  PEPTIC  DIGESTION. 

One  hundred  grammes  of  moist  fibrin  that  has  been  thoroughly 
washed  in  running  water  are  placed  in  1000  c.c.  of  an  0.3  per  cent, 
solution  of  hydrochloric  acid,  to  which  a  few  grammes  of  pepsin 
have  been  added.  The  mixture  is  kept  at  a  temperature  of  40°  C, 
and  may  be  examined  for  the  primary  products  of  digestion  after 
about  two  hours,  while  the  secondary  products  can  only  be  demon- 
strated after  a  longer  period  of  time.  Fr(jm  time  to  time  it  is 
then  necessary  to  ascertain  whether  free  hydrochloric  acid  is  still 
present,  and  to  add  an  additional  amount  whenever  it  is  wanting, 
so  that  the  acidity  referable  to  free  acid  remains  about  the  same 
during  the  entire  period  of  digestion.  The  occasional  addition  of 
a  little  pepsin  is  also  advisable.  The  first  specimen  for  examina- 
tion is  taken  after  two  hours.  The  liquid  is  filtered  and  neutral- 
ized with  a  dilute  solution  of  sodium  hydrate.  The  precipitate 
which  thus  forms  consists  of  syntonin  and  is  filtered  off.  The  fil- 
trate is  rendered  feebly  acid  with  very  dilute  acetic  acid,  treated 
with  an  equal  volume  of  a  saturated  solution  of  common  salt,  and 
boiled.  Any  native  coagulable  albumin  that  may  be  present  is 
thus  precipitated  and  is  filtered  off  on  cooling.  The  solution  is 
again  made  neutral  and  treated  with  an  equal  volume  of  a  satu- 
rated solution  of  ammonium  sulphate.  In  this  manner  the  primary 
albumoses  of  Kiihue  are  precipitated,  and  are  filtered  off  after  stand- 
ing for  about  one-half  hour.  To  separate  the  proto-albumose  froin 
the  he'ero-albumose,  the  precipitate  is  dissolved  in  hot  water  and 
treated  with  an  equal  volume  of  95  per  cent,  alcohol.  On  standing, 
the  hetero-albumose  separates  out,  wliile  the  proto-albumose  is  found 
in  the  alcoholic  filtrate.  It  is  purified  by  repeated  solution  in  hot 
water  and  precipitation  with  alcohol.  To  isolate  the  proto-albumose, 
the  alcoholic  filtrate  is  evaporated  to  dryness  on  a  water-batli,  or  the 
alcohol  is  distilled  off'  in  the  vacuum.  The  remaining  material  is 
repeatedly  dissolved  in  water  and  treated  with  alcohol  until  the 
alcoholic  solution  remains  clear  on  standing.  To  this  end,  it  is 
usually  necessary  to  repeat  the  solution  in  water  and  the  treatment 
with  alcohol  five  or  six  times. 

198 


THE  PRODUCTS   OF  PEPTIC  DIGESTION.  199 

The  further  steps  in  the  digestion  of  fibrin  may  be  studied  in  a 
specimen  which  has  been  kept  at  a  temperature  of  40°  C.  for  two 
to  three  weeks.  Syntonin  or  native  sohible  albumin  that  may  still 
be  present,  as  well  as  the  primary  albumoses,  are  removed  as  just 
described.  The  neutral  solution  is  then  treated  with  one-half  its 
volume  of  a  saturated  solution  of  ammonium  sulphate.  In  this 
manner  a  two-thirds  saturation  of  the  solution  is  eftected,  and  on 
standing  the  deutero-fraction  A  separates  out;  this  is  filtered  off,  and 
the  solution  saturated  with  ammonium  sulphate  in  substance.  Asa 
result  the  deutero-fraction  B  is  thrown  down,  and  on  acidi- 
fying the  filtrate  with  one-tenth  its  volume  of  a  solution 
of  sulphuric  acid  that  has  been  saturated  with  ammt)nium  sul- 
phate, and  of  which  10  c.c;  correspond  in  strength  to  17  c.c.  of 
a  one-tenth  normal  solution  of  sodium  hydrate,  the  deutero-albu- 
mose-C  finally  separates  out  on  standing.  The  resulting  filtrate  is 
then  free  from  albumoses,  and  should  contain  the  amphopeptone  of 
Kiihne.  But,  as  I  have  indicated,  two  additional  fractions  can  be 
obtained  from  the  final  solution.  To  this  end,  a  solution  of  iodo- 
potassic  iodide,  containing  two  parts  of  the  iodide  to  one  part  of 
iodine,  which  has  been  saturated  with  ammonium  sulphate,  is  added 
until  precipitation  is  complete.  The  material  which  is  thus  thrown 
down  is  placed  in  96  per  cent,  alcohol.  Peptone-B  then  passes  into 
solution,  while  peptone-A  remains  undissolved.  This  portion  is  dis- 
solved in  a  little  warm  water,  the  solution  saturated  with  ammonium 
sulphate,  and  reprecipitated  with  the  iodine  solution.  The  peptone 
is  then  redissolved  in  Marm  water,  reprecipitated  with  alcohol,  and 
freed  from  any  remaining  iodine  by  shaking  with  ether.  Peptone-B, 
on  the  other  liand,  is  obtained  by  evaporating  its  alcoholic  solution 
to  dryness,  when  the  residue  is  dissolved  in  water  and  freed  from 
iodine  by  shaking  Avith  ether. 

THE   PRODUCTS    OF   TRYPTIC   DIGESTION. 

One  hundred  grammes  of  moist  fibrin,  as  in  the  above  experi- 
ments, are  placed  in  a  liter  of  an  0.25  per  cent,  solution  of  sodium 
carbonate,  to  which  a  few  grammes  of  commercial  pancreatin  have 
been  added.  Putrefaction  is  guarded  against  by  the  addition  of 
chloroform  and  thymol.  Tlie  mixture  is  kept  at  a  temperature  of 
40°  C,  and  can  be  examined  after  twenty-four  to  thirty-six  hours. 
For  the  preparation  of  antipeptone,  however,  in  amounts  which 
can  be  utilized  to  demonstrate  the  presence  of  the  hexon  bases,  it 
is  necessary  to  take  a  much  larger  quantity  of  fibrin  and  to  extend 
the  period  of  digestion  over  several  weeks.  From  1410  grammes 
Kutscher  claims  to  have  obtained  as  much  as  200  grammes. 

The  filtered  fluid  is  first  neutralized  with  dilute  sulphuric  acid,- 
which  causes  the  separation  of  any  alkaline  alliuminate  that  may  be 
present.  Coagulable  albumins  are  removed  by  acidifying  the  solu- 
tion with  acetic  acid  and  boiling.     The  solution  is  then  treated  with 


200  THE  PRODUCTS  OF  ALBUMINOUS  DIGESTION. 

one  and  one-half  times  its  volume  of  a  saturated  solution  of  ammo- 
nium sulpiiate.  On  standing,  the  deutero-fraction  A  separates  out. 
On  complete  saturation  witii  the  salt  in  substance  the  deutero-frac- 
tion B  is  obtained  ;  a  C-albumose  is  not  formed  on  tryptic  digestion. 
On  acidifying  with  sulphuric  acid,  however,  as  in  the  study  of  peptic 
digestion,  it  may  happen  that  a  turbidity  appears,  which  is  prol)ably 
due  to  the  presence  of  Xeumeister's  antideutero-albimiose,  resulting 
from  anti-albumid.  The  tinal  filtrate  then  contains  the  common 
amido-acids,  antipeptone,  tryptophan,  etc. 

Reactions  of  the  Individual  Albumoses. 

Whether  or  not  the  different  albumoses  which  are  formed  during 
the  process  of  digestion  are  qualitatively  the  same,  irrespective  of 
their  origin,  is  not  known,  but  it  is  likely  that  certain  differences 
exist.  Quantitative  differences  also  undcjubtedly  occur,  as  is  suggested 
a  priori  by  the  varying  amounts  of  the  individual  end-products 
Avhieh  can  be  obtained  on  hydrolysis.  The  following  account  of  the 
individual  albumoses  is  largely  based  on  a  study  of  the  fibrinoses. 

Hetero-albumose. — Hetero-albumoseis  more  closely  related  to  the 
oriuinal  albuminous  molecule  than  any  other  albuniose.  It  is  but 
little  soluble  in  water  and  does  not  dialyze  in  neutral  solution.  On 
heating  a  fairly  concentrated  solution  in  the  presence  of  a  moderate 
amount  of  salt,  partial  coagulation  occurs  between  55°  and  60°  C.  ; 
on  further  heating,  partial  solution  takes  j)lace.  On  standing 
hetero-albumose  becomes  insoluble  in  part  (Kiihne's  dysalbumose) ; 
but  u]>on  the  addition  of  a  small  amount  of  soda  redissolution  occurs 
and  hetero-albumose  again  results.  The  same  occurs,  when  the  heat 
coagulum  is  dissolved  in  dilute  hydrochloric  acid.  A  denaturiza- 
tion  thus  does  not  occur.  In  acid  solution  hetero-albumose  is  pre- 
cipitated in  tofo  on  half-saturation  with  sodium  chloride.  Its  limits 
of  precipitation  for  ammonium  sulphate  in  neutral  solution  are  2.6 
and  4.4.  Alcohol  ])recipitates  it  very  readily  ;  it  is  insoluble  in  the 
presence  of  32  per  cent.  With  the  usual  albumin  precipitants 
hetero-albumose  shows  a  typical  albumose  reaction  (page  63).  Its 
elementary  composition  'is  C  =  55.12,  11  =  6.61,  N  =  17.P8, 
S  =  1.22,  0=19.07.  It  contains  no  carbohydrate  grouj).  aha 
possibly  also  no  tyrosin  or  tryj^tophan  radicle  ;  it  accordingly  does 
not  give  the  Molisch  reaction  or  that  of  Adamkiewicz.  The  sub- 
stance is  an  anti-l)odv  in  the  sense  of  Kiihne,  and  is  accordingly 
related  to  Kiihne's  anti-albumid  and  the  antipeptone  group.  On 
further  digestion  it  yields  deutero-albumoses  of  tiie  A  and  B  group, 
traces  of  deutero-albumose  C,  and  a  considerable  amount  of  Pick's 
peptone  B.  On  hydrolysis  it  yields  much  leucin  and  glycocoll  and 
39  per  cent,  of  the  total  nitrogen  in  basic  form  ;  it  also  contains  the 
phenyl-alanin  com]ilex. 

Proto-albumose. — Proto-albumose  in  its  physical  behavior  sliows 
that  it  stands  further  removed  from  the  original  albuminous  mole- 


THE  PRODUCTS  OF  TRYPTIC  DIGESTION.  201 

cule  than  hetero-albumose.  It  is  quite  readily  soluble  in  water  and 
diffuses  to  some  extent  through  vegetable  parchment.  It  is  not 
coagulated  by  heat.  With  nitric  acid  only  partial  precipitation 
occurs.  The  alkaloidal  reagents  cause  the  precipitation  of  proto- 
albumose,  but  the  precipitate  redissolves  in  an  excess  ot"  the  reagents. 
Sodium  chloride  brings  about  complete  precipitation  only  in  feebly 
acid  solution,  on  complete  saturation.  With  ammonium  sulphate 
the  limits  of  precipitation  are  the  same  as  for  the  hetero-albumose. 
In  dilute  alcohol  proto-albumose  dissolves  with  ease ;  preci])itation 
begins  in  the  presence  of  80  per  cent,  (separation  from  hetero-albu- 
mose), and  only  becomes  complete  when  a  mixture  of  alcohol  and 
ether  is  added. 

The  elementary  composition  is  C=  55.64,  H  =  6.8,  N=  17.66, 
S  =  1.21,  O  =  18.69.  It  is  a  hemi-body  and  accordingly  readily 
digested  further;  it  yields  much  deutero-albumose  of  the  A  fraction, 
some  deutero-albumose  B  and  peptone  B,  but  no  deutero-albumose 
C  or  peptone  A.  Like  hetero-albumose,  it  contains  no  carbohydrate 
group  and  hence  does  not  give  the  Molisch  reaction.  On  hydrolysis 
it  yields  25  per  cent,  of  its  nitrogen  in  basic  form,  much  tyrosin  and 
tryptophan,  but  little  leucin  and  glycocoll. 

Tlie  remaining  albumoses  are  not  yet  known  so  well  as  the  two 
that  have  been  described  ;  the  most  noteworthy  data  follow  : 

Gluco-albumose. — The  gluco-albumose  (synalbumose)  is  ob- 
tained together  with  the  deutero-fraction  B  on  complete  saturation 
with  ammonium  sulphate,  the  reaction  being  neutral.  It  can  be 
se])arated  from  the  deutero-fraction  B  by  careful  treatment  with 
alcohol  (see  below).  Its  elementary  composition  is  C=  48.72, 
H  =  7.03,  N  =  13.61-14.77,  S  +  O  =  30.49.  There  is  but  little 
loosely  combined  sulphur.  The  substance  gives  an  intense  Molisch 
reaction,  and  is  the  only  primary  albumose  containing  a  carbohy- 
drate group.  This  can  be  split  off  by  means  of  acids,  and  yields  an 
osazon,  which  in  appearance  resembles  glucosazon  and  melts  between 
182°  and  184°  C'.  On  further  digestion  it  yields  Pick's  peptone  A  ; 
the  intermediate  ]>roducts  have  not  yet  been  isolated. 

Deutero-fraction  A. — The  deutero-fraction  A  is  precipitated  on 
62  per  cent,  saturation  with  ammonium  sulphate.  By  treating  this 
fraction  with  60-70  per  cent,  alcohol  it  can  be  further  resolved  into 
two  fractions,  of  which  the  one  (thio-albumose)  is  precipitated,  while 
the  other,  deutero-albumose  A,  passes  into  solution.  The  tliio- 
albumose  derives  its  name  from  the  large  amount  of  sulphur  which 
it  contains,  viz.,  2.97  per  cent.,  and  which  is  present  almost  entirely 
in  loosely  combined  form  (cvstin).  The  elementary  composition  of 
this  portion  is:  C  =  48.96,  H  =  6.9,  N=  16.02,  S  :- 2.97, 
O  =  25.1 5. 

The  deutero-albumose  A  contains  much  less  sul])hur,  which  is 
also  present  in  loosely  combined  form.  Its  composition  is  C  =  53.11, 
H  =  7.16,  N  =  17.86,  S  =  0.8,  and  0  =  21.07. 

Neither  fraction  probably  contains  the  carbohydrate  group,  while 


202 


THE  PRODUCTS  OF  ALBUMINOUS  DIGESTION. 


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204  THE  PRODUCTS   OF  ALBUMINOUS  DIGESTION. 

the  Millon  reaction,  the  xanthoproteic  and  biuret  reactions  are  posi- 
tive.    On  fusion  with  caustic  alkali  indol  is  obtained. 

Deutero-fraction  B. — This  fraction  is  obtainetl  together  with  the 
gluco-all)uniose  on  complete  saturation  with  ammonium  sulphate  in 
neutral  solution.  It  consists  of  two  fractions,  which  can  be  differ- 
entiated from  each  other  and  the  gluco-albumose  by  means  of  alco- 
hol. 35  per  cent,  alcohol  will  precipitate  the  deutero-albumose  B ; 
the  gluco-all)uniose  is  then  thrown  down  in  the  presence  of  60  to  70 
per  cent.  ;  while  the  B  fraction  remains  soluble  even  with  80  per 
cent.  Deutero-albumose  B,  in  the  case  of  fibrin  at  least,  is  formed 
only  in  traces,  and  may  indeed  be  absent.  It  does  not  give  the  re- 
action of  Molisch  ;  tlie  biuret  reaction  is  intense  and  the  sul]>hur 
reaction  marked.     It  contains  16.94  per  cent,  of  nitn^gen. 

Deutero-albumose  B. — This  usually  represents  the  greater  por- 
tion uf  the  secondary  albumoses.  It  is  not  absolutely  insoluble  in 
alcohol,  but  a  large  excess  of  strong  alcohol  is  necessary  to  ]>recipi- 
tate  it  from  a  concentrated  solution.  The  substance  does  not  give 
Molisch's  reaction,  nor  does  it  contain  loosely  combined  sulj)hur  ; 
the  biuret,  xanthoproteic,  and  Millon's  reactions,  however,  are  ])res- 
ent  and  the  indol  reaction  is  very  pronounced.  Elementary  analysis 
of  this  portion  has  not  given  constant  results,  which  suggests  that 
the  substance  as  obtained  by  Pick  was  not  pure.  He  found  as  a 
matter  of  fact  that  his  material,  which  was  obtained  from  Witte 
peptone,  was  contaminated  by  a  melanin-like  body,  which  he  termed 
peptomekinm.  The  analytical  figures  follow:  0  =  43.99-52.32, 
H  =  6.91-7.32,  N  =  1 4.25-1 5.38,  S  =  1 .63-1.21 ,  O  =  33.23-23.79. 

Deutero-fraction  C. — This  is  precipitated  on  complete  saturation 
wnth  ammonium  suljdiate,  and  carefully  acidifying  with  sul])huric 
acid  saturated  with  ammonium  sulphate.  It  is  soluble  in  76-80  per 
cent,  alcohol.  In  the  case  of  fibrin  deutero-albumose  C  is  formed 
only  in  small  amount.  It  contains  no  loosely  combined  sulphur 
and  does  not  give  the  reaction  of  Molisch  nor  that  of  Millon,  or  to 
so  slight  a  degree  only  as  to  suggest  contamination  with  other  frac- 
tions. On  fusion  with  caustic  alkali  it  yields  neither  indol  nor 
skatol.  Elementary  analysis  has  given  the  following  results  : 
0  =  34.52,  H  =  5.35,  N  =  17.24. 

The  fraction  apparently  represents  an  end-product  of  peptic  di- 
gestion ;  it  yields  no  peptone  (Pick).  It  is  not  formed  during 
tryptic  digestion.  The  specific  reactions  of  the  individual  fractions 
are  given  in  the  accompanying  tables  (pages  202  and  203). 

THE  END-PRODUCTS  OF  ALBUMINOUS  DIGESTION. 

To  study  the  end-products  of  albuminous  digestion  it  is  well  to 
digest  fibrin  for  several  weeks  with  trypsin  as  just  described.  Any 
deutero-albumoses  still  remaining  are  removed  and  the  antipeptone 
fraction  isolated  as  follows  : 

Antipeptone  Fraction. — The  mixture  is  filtered,  slightly  acidified 


THE  END-PRODUCTS  OF  ALBUMINOUS  DIGESTION.      205 

with  acetic  aciJ,  boiled,  again  filtered,  and  concentrated  to  about 
1000  c.c.  On  cooling,  a  good  deal  of  tyrosin  separates  out  and  is 
filtered  off.  The  filtrate  is  diluted  with  water  to  about  2000  c.c, 
neutralized,  heated  to  near  the  boiling-point,  and  saturated  with 
ammonium  sulphate  in  substance.  On  cooling,  any  albumoses  that 
may  have  separated  out,  together  witli  a  large  quantity  of  the  salt,  are 
filtered  off.  The  filtrate  is  heated,  and,  while  boiling,  rendered  strongly 
alkaline  with  ammonia  and  ammonium  carbonate,  and  again  saturated 
with  ammonium  sulphate.  On  cooling,  a  second  fraction  of  albu- 
moses is  filtered  off.  The  solution  is  then  heated  until  the  odor  of 
ammonia  has  disappeared;  ammonium  sulpiiate  is  again  added  to 
saturation,  and  the  liquid  rendered  distinctly  acid  with  acetic  acid, 
when  on  cooling  a  third  fraction  of  albumoses  separates  out  and  is 
filtered  off.  The  filtrate  is  concentrated  to  about  one  liter  and  freed 
from  a  large  amount  of  ammonium  sulphate,  which  separates  out  on 
cooling.  It  is  then  diluted  with  water  to  about  3000  c.c,  and  treated 
at  a  temperature  of  30°  C.  with  barium  hydrate  in  substance,  to  re 
move  the  remaining  salt.  A  slight  excess  of  the  barium  is  removed 
with  carbonic  acid,  and  is  filtered  off  after  boiling  for  a  moment.  The 
filtrate  is  evaporated  to  about  1000  c.c,  when  the  barium  peptone  is 
decomposed  with  dilute  sulphuric  acid,  care  being  taken  that  the 
acid  is  not  added  in  excess.  The  resulting  barium  sulphate 
is  filtered  off  and  the  filtrate  concentrated  to  a  thin  syruji.  On  cool- 
ing, absolute  alcohol  is  added  until  the  turbidity  that  first  a]ipears 
no  longer  disappears  on  stirring.  After  filtering  with  the  aid  of  a 
suction  pump,  the  solution  is  poured  into  absolute  alcohol  while 
stirring.  The  antipeptone  is  then  precipitated  and  allowed  to  settle, 
when  the  supernatant  fluid  is  siphoned  off  and  the  antipeptone  col- 
lected on  a  filter  with  the  aid  of  a  suction-pump.  It  is  finally 
washed  with  absolute  alcohol,  then  with  ether,  and  rapidly  placed  in 
a  desiccator  over  sulphuric  acid. 

From  this  material  the  three  hexon  bases  may  be  isolated,  as 
described  by  Kutscher  (Zcit.f.  phys.  Chem.,  1898,  vol.  xxvi.,  p.  110). 

The  Mono-amido-acids.'^To  study  the  mono-amido-acids  the 
antipeptone  fraction  need  not  be  removed.^ 

Leucin. — Aside  from  its  formation  during  the  process  of  pancreatic 
digestion  or  on  artificial  decomposition  of  albumin  with  dilute  mineral 
acids  and  alkalies,  leucin  has  been  demonstrated  in  the  spleen,  in  the 
lymph-glands,  in  the  thyroid,  the  kidneys,  the  liver,  and  the  brain, 
though  mostly  under  pathological  conditions,  when  it  may  also 
appear  in  the  urine.  It  is  further  found  in  sheep's  wool,  in  decom- 
posing epithelial  structures,  as  in  the  desquamated  material  which  is 
found  between  the  toes,  etc.  Its  presence  in  the  intestine  may  also 
be  due  to  the  action  of  bacteria  upon  the  albuminous  products  of 
digestion. 

In  pure  form  leucin  crystallizes  in   extremely  thin  white  lustrous 

>  For  the  isolation  of  the  various  mono-ainido-acids,  according  to  E.  Fischer's  method, 
see  Zeit.  f.  phys.  Chem.,  1901,  vol.  xxxiii.,  p.  151. 


206  THE  PRODUCTS  OF  ALBUMINOUS  DIGESTION. 

platelets  ;  but  more  commonly  it  is  seen  in  the  form  of  spherules  of 
variable  size,  which  closely  resemble  globules  of  fat.  In  these,  con- 
centric striatious,  as  well  as  very  fine  radiating  lines,  can  at  times  be 
made  out  on  careful  examination. 

Several  leucins  apparently  exist.  One  form,  which  can  be  pro- 
duced synthetically  from  hydrocyanic  acid  and  ammonium-iso- 
valerianic  aldehyde,  is  optically  inactive.  The  common  leucin, 
on  the  other  hand,  which  is  formed  during  tryptic  digestion  or  on 
decomposition  of  the  native  albumins  with  hydrochloric  acid,  is 
dextrorotatory.  A  third  form  results  from  the  action  of  Penicilinm 
glaucum  upon  the  inactive  substance,  and  is  said  to  be  Isevorotatory. 
According  to  Cohn,  moreover,  several  isomeric,  optically  active 
leucins  exist.  The  common  form  is  easily  soluble  in  water,  in 
alkalies  and  acids,  as  also  in  hot  alcohol  ;  in  ether  it  is  insoluble. 
It  combines  with  acids,  alkalies,  and  the  oxides  of  some  of  the  heavy 
metals  to  form  salts.  On  boiling  a  solution  of  leucin  with  subacetate 
of  lead  the  corresponding  compound  of  lead  oxide  can  thus  be 
obtained  if  ammonia  is  carefully  added  to  the  cooled  solution.  A 
copper  salt  is  similarly  formed  if  leucin  in  aqueous  solution  and  con- 
taining a  small  amount  of  alkali  is  treated  with  a  solution  of  cupric 
sulphate,  care  being  taken  not  to  add  an  excess.  On  standing,  the 
compound  separates  out  in  the  form  of  clusters  of  blue  needles,  which 
are  cliaracterized  by  their  pronounced  insolubility. 

AVhen  carefully  heated  to  a  temperature  of  170°  C.  leucin  melts 
and  sublimes  in  the  form  of  white  flakes,  which  are  deposited  on  the 
cooler  portion  of  the  tube.  At  the  same  time  the  odor  of  amylamin 
develops. 

On  evaporating  a  small  amount  of  leucin  upon  platinum  foil  with 
nitric  acid  a  colorless  residue  is  formed.  If  to  this  a  drop  of  sodium 
hydrate  solution  is  added  and  heat  is  carefully  applied,  a  yellowish 
or  brownish  color  develops,  and  on  further  heating  an  oil-like  droplet 
is  obtained,  which  rolls  about  upon  the  platinum  without  adhering 
(Scherer's  test). 

On  decomposition  with  an  alkali  or  during  the  process  of  putre- 
faction leucin  yields  ammonia  and  valerianic  acid.  On  oxidation 
leucinic  acid  results. 

As  has  been  indicated,  leucin  is  an  amido-capronic  acid  of  the 
formula  (CH3)2.CH.CH2.CH(NH2).COOH,  and  may  hence  also  be 
regarded  as  «-amido-isobutyl-acetic  acid. 

Tyrosin. — Tyrosin  can  be  obtained  on  tryptic  digestion  from  all 
those  albumins  in  which  aromatic  groups  exist.  Collagen,  in  which 
this  is  absent,  accordingly  yields  no  tyrosin,  and  very  small  amounts 
only  are  obtained  from  elastin.  In  the  animal  body  it  is  practically 
found  as  such  only  under  pathological  conditions  if  we  disregard  the 
minute  quantity  which  is  formed  in  the  intestinal  canal.  Like 
leucin,  it  is  also  formed  during  the  process  of  all)uminoiis  putrefaction, 
and  can  ])e  obtained  artificially  by  decomposing  albuminous  sub- 
stances with  dilute  mineral  acids  or  alkalies. 


THE  END-PRODUCTS  OF  ALBUMINOUS  DIGESTION.       207 

While  impure  ty rosin  may  occur  in  the  form  of  spherules  similar 
to  those  of  leucin,  the  pure  substance  crystallizes  in  delicate,  silky 
needles,  which  are  often  grouped  m  sheaves  and  rosettes.  Accord- 
ing to  its  mode  of  formation,  the  substance  is  optically  inactive,  as 
when  ibrmed  synthetically  or  by  decomposition  with  baryta-water, 
or  it  is  Isevorotatorv  when  derived  from  albumins  on  boiling  with 
acids.  In  cold  water  it  is  only  slightly  soluble,  while  in  boiling 
water  it  dissolves  in  the  proportion  of  1  to  154.  Its  solubility  is 
increased  in  the  presence  of  alkalies  or  mineral  acids.  In  alcohol 
and  ether  it  is  insoluble. 

Tvrosin  may  be  regarded  as  para-oxv-phenvl-propionic  acid,  and 
has  the  formula  C,H,(bH).CH,.CH(NH2).C06H.  It  may  be  tbrmed 
synthetically  from  ethylene  oxide  and  para-amido-benzoic  acid,  and 
can  also  be  obtained  from  para-amido-phenyl  alanin  and  para-nitro- 
phenyl  alanin.  On  bacterial  decomposition  it  yields  hydroparacu- 
maric  acid  (para-oxy-phenyl-propionic  acid),  which  can  be  further 
transformed  into  para-oxy-phenyl-acetic  acid,  and  this  into  para- 
c  resol,  as  has  been  shown  (page  S9.)  On  fusion  with  caustic  alkali, 
an  the  other  hand,  it  gives  rise  to  the  formation  of  pa ra-oxy -benzoic 
;;cid,  acetic  acid,  and  ammonia,  as  is  shown  in  the  equation  : 

OH  /OH 

CgHi  +  H^O  +  O  =  CgH^/  +  CH3.COOH  +  NH3. 

-CH2.CH(NH,).C00H  ^COOH 

Oil  oxidation  with  potassium  bichromate  and  sulphuric  acid  hydro- 
cyanic acid,  benzoic  acid,  acetic  acid,  and  formic  acid  result. 

With  acids  and  alkalies,  as  also  with  certain  salts  of  the  heavy 
metals,  tvrosin  combines  with  difficulty  to  form  salt-like  bodies. 

Tests  for  Tyrosin. — Hoffmann's  Test. — With  Millon's  reagent 
t^  rosin  gives  the  well-known  reaction  of  those  albumins  in  which 
aromatic  groups  are  present,  but,  as  would  be  expected,  in  a  degree 
n;uch  more  intense.  The  reaction  is  due  primarily  to  the  formation 
o*"  oxy-benzoic  acid  (salicylic  acid). 

Piria's  Test. — A  few  crystals  of  tyrosin  are  dissolved  in  con- 
contrated  sulphuric  acid  and  heated  to  about  100°  C,  when  the  sub- 
stance dissolves.  Tyrosin-sulphuric  acid  is  thus  formed,  and  gives 
rise  to  a  red  color.  On  cooling,  the  liquid  is  diluted,  and  treated  with 
barium  carbonate  while  heating  until  the  reaction  becomes  just 
alkaline.  Tyrosin-sulphonate  of  barium  thus  results,  which  gives 
rise  to  a  dark-violet  color  on  treating  with  a  very  dilute  solution  of 
sesqnichloride  of  iron.  An  excess  of  iron,  however,  must  be  care- 
fully avoided.     Oxy-benzoic  acid  gives  the  same  reaction. 

Scherer's  Test. — On  evaporating  tyrosin  Avith  a  few  drops  of 
nitric  acid  on  platinum  foil  a  yellow,  transparent  residue  is  obtained, 
which  turns  red  on  moistening  the  substance  with  a  drop  of  sodium 
hydrate  solution,  and  becomes  brown  on  further  evaporation.  The 
reaction  is  due  to  the  formation  of  nitro-tyrosin  nitrate,  but  is  not 
characteristic,  as  other  bodies  behave  in  a  similar  manner. 


208  THE  PRODUCTS  OF  ALBUMINOUS  DIGESTION. 

Momer's  Test. — A  small  amount  of  tyrosin,  in  substance  or  in 
solution,  is  addctl  to  a  few  c.e.  of  a  special  reagent  which  has  the 
composition:  formalin,  1  ])art ;  distilled  water,  45  parts;  and  con- 
centrated sulphuric  acid,  bb  parts.  The  mixture  is  boiled,  when 
either  at  once  or  a  few  minutes  after  boiling  has  begun  a  fine  green 
color  develops  which  persists  for  a  long  time.  Other  compounds 
which  are  closely  related  to  tyrosin,  as  also  native  albumins,  albu- 
moses,  and  albuminoids,  do  not  give  the  reaction.  ^Vith  chemically 
pure  7J-oxyphenyl  propionic  acid  and  the  corresponding  acetic  acid 
Morner  had  not  been  aljle  to  test  his  reaction. 

Isolation  of  Leucin  and  Tjrrosin. — To  isolate  leucin  and  tyrosin 
among  the  final  products  of  tryptic  digestion,  and  to  separate  the 
two  from  each  other,  the  digestive  mixture  is  first  freed  from  alkaline 
albuminate,  coagulable  albumins,  and  the  albumoses,  as  already 
described  (page  199).  The  final  filtrate  is  concentrated  to  a  syrupy 
consistence,  when  on  cooling  leucin  and  tyrosin  crystallize  out.  The 
mass  of  crystals  is  then  boiled  with  a  large  quantity  of  water,  to 
wdiich  a  sufficient  amount  of  ammonia  is  added  to  insure  solution  of 
the  substances.  The  boiling  solution  is  treated  with  subacetate  of 
lead  until  the  resulting  precipitate  appears  almost  white.  The  fil- 
trate is  brought  to  the  boiling-point,  neutralized  with  sulphuric  acid, 
and  filtered  while  boiling  hot.  On  cooling,  the  tyrosin  crystallizes 
out,  while  the  leucin  remains  in  solution.  The  former  can  then  be 
purified  by  recrystallization  from  boiling  water  or  from  very  dilute 
ammonia.  The  solution  which  contains  the  leucin  is  freed  from  lead 
with  hydrogen  sulphide,  and  the  filtrate  is  concentrated  and  boiled 
with  an  excess  of  freshly  precipitated  cupric  hydrate.  A  portion  of 
the  leucin  is  thus  precipitated,  while  the  rest  remains  in  solution, 
but  partly  crystallizes  out  on  cooling  as  the  corresponding  copper 
compound.  The  precipitate  is  placed  in  the  copper-containing 
solutif)n,  and  is  freed  from  copper  with  hydrogen  sulphide  ;  the 
filtrate  is  then  decolorized  M"ith  animal  charcoal,  strongly  concen- 
trated, and  set  aside  for  crystallization. 

Asparaginic  Acid. — While  asparaginic  acid  is  apparently  formed 
from  all  albuminous  substances  on  digestion  with  trypsin,  the  largest 
amounts  are  obtained  from  fibrin  and  gelatin.  Like  leucin  and 
tvrosin,  it  likewise  results  on  artificial  decomposition  of  the  albu- 
mins Avith  dilute  mineral  acids  and  alkalies,  and  is  also  formed 
during  the  process  of  albuminous  putrefaction.  Outside  the  in- 
testinal canal  asparaginic  acid  has  not  been  found  in  the  animal 
body.  In  the  form  of  its  amide  asparagin,  however,  it  is  widely 
distributed  in  the  vegetable  world,  and  supposedly  plays  an  im- 
portant role  in  the  synthesis  of  the  vegetable  albumins. 

The  substance  crystallizes  in  rhombic  prisms,  which  are  soluble 
with  difficulty  in  cold  water,  but  are  quite  soluble  in  hot  water.  In 
absolute  alcohol  it  is  insoluble.  Its  aqueous  solutions  are  laevorota- 
tory,  while  in  the  presence  of  nitric  acid  dextrorotation  is  observed. 

As  has  been  shown  (page  88),   asparaginic  acid  is  a  dibasic  acid 


THE  END-PRODUCTS  OF  ALBUMINOUS  DIGESTION.       209 

of  the  fatty  series.  It  is  amido-suceinic  acid,  and  is  represented  by 
the  formula  CH,.CI-I(NHo).(COOH),.  It  can  be  obtained  from 
asparagin  on  boiling  with  hydrochloric  acid,  as  shown  in  the 
equation  : 

CH2.CONH2  CH2.COOH 

I  +  HjO  =  I  -I-  NHo. 

CH(NH2).C00H  CH(NH,).COOH 

Asparagin.  Asparaginic  acid. 

It  has  also  been  produced  synthetically.  On  reduction  it  yields 
succinic  acid,  as  has  been  shown. 

With  cupric  oxide  asparaginic  acid  forms  a  crystalline  compound 
which  is  almost  insoluble  in  cold  water,  but  dissolves  in  boiling 
water  with  comparative  ease.  This  property  is  utilized  for  the  pur- 
pose of  isolating  the  substance  from  the  mixture  of  digestive 
products. 

Glutaminic  Acid. — Whether  or  not  glutaminic  acid  is  formed 
during  the  tryptic  digestion  of  the  albumins  in  general  has  not  as 
yet  been  ascertained.  Kutscher  claims  to  have  found  it  in  the 
so-called  antipeptone  of  Kiihne,  which  was  obtained  from  fibrin. 
On  boiling  with  strong  mineral  acids,  however,  it  is  constantly 
formed.  But  it  is  noteworthy  that  much  larger  quantities  are  found 
if  the  decomposition  of  the  albumins  is  eifected  with  hydrochloric 
acid  than  with  sulphuric  acid.  Kutscher  thus  found  only  1.8  per 
cent,  among  the  decomposition-products  of  casein  when  using  sul- 
phuric acid,  while  Hlasiwez  and  Habermann  obtained  as  much  as 
29  per  cent,  when  hydrochloric  acid  was  used.  This  is,  of  course, 
remarkable,  and  it  would  be  exceedingly  interesting  to  ascertain  the 
fate  of  those  radicles  which  can  yield  so  large  an  amount  of  gluta- 
minic acid  when  decomposition  is  effected  by  hydrochloric  acid. 

Glutaminic  acid  crystallizes  in  small  glistening  crystals,  which 
are  soluble  with  difficulty  in  cold  water,  while  in  boiling  water  they 
dissolve  with  greater  ease,  but  separate  out  on  cooling.  With  acids 
and  alkalies  it  combines  to  form  salt-like  products,  among  which  the 
hydrochlorate  is  conveniently  utilized  for  the  purpose  of  identifying 
the  substance.     The  melting-point  of  this  compound  is  193°  C 

The  composition  of  glutaminic  acid  is  expressed  by  the  formula 
CH..CH,.CH(NH,).(COOH),.  It  is  thus  amido-glutaric  acid,  and 
bears  the  same  relation  to  glutamin  as  that  which  exists  between 
asparaginic  acid  and  asparagin.     This  is  represented  in  the  equation  : 

/CONH2  /COOH 

CH2.CH2.CH(NH,)<  +   HP   =   CH^.CH^.CHfNH,)/  +  NHo^ 

\COOH  \COOH 

Glutamin.  Glutaminic  acid. 

On  reduction  it  yields  glutaric  acid. 

Isolation  of  Asparaginic  Acid  and  Glutaminic  Acid. — To  isolate  the 
two  acids  in  question  among  the  products  of  tryptic  digestion,  the 
mixture  must  first  be  freed  from  albumins  and  albumoses,  as  has 
been  described.     The  remaining  solution  is  acidified  with  sulphuric 

14 


2i0  THE  PRODUCTS  OF  ALBUMINOUS  DIGESTION. 

acid  and  precijiitated  with  phosphotungstic  acid.  The  filtrate  is 
freed  from  sulphuric  acid  and  any  excess  of  the  phosphotuno-stic 
acid  by  means  of  barium  hydrate.  From  the  resultinoc  filtrate 
leucin  and  tyrosin  are  then  removed  by  concentration.  The  mother- 
liquor  contains  the  glutaminic  acid  and  asparaginic  acid.  These 
are  now  separated  from  each  other  in  the  following  manner :  the 
diluted  solution  is  brought  to  the  boiling-point  and  digested  with 
carbonate  of  copper.  It  is  filtered  while  still  hot,  and  precipitated 
with  subacetate  of  lead,  care  being  taken  to  avoid  an  excess.  This 
precipitate  is  decomposed  with  hydrogen  sulphide,  and  the  filtrate 
concentrated  to  a  small  volume.  On  standing,  a  crystalline  mass  is 
obtained,  which  is  then  dissolved  in  boiling  water  and  digested  wnth 
an  excess  of  carbonate  of  copper,  as  before.  The  hot  filtrate  is 
again  concentrated,  when  on  standing  the  copper  salt  of  asparaginic 
acid  separates  out  in  characteristic  groups  of  needles.  The  filtrate 
is  freed  from  copper  by  means  of  hydrogen  sulphide,  concentrated^ 
and  set  aside,  wdien  the  glutaminic  acid  crystallizes  out. 

GlycocoU. — AVhile  it  is  generally  known  that  glycocoll  plays  an 
important  part  in  the  nitrogenous  metabolism  of  the  animal  body^ 
and  is  intimately  concerned  in  the  formation  of  urea,  hippuric  acid, 
phenaceturic  acid,  certain  biliary  acids,  and  in  birds  and  reptiles  of 
uric  acid,  it  is  of  interest  to  note  that  the  substance  has  thus  far 
not  been  found  as  such  among  the  products  of  pancreatic  digestion, 
although  its  radicle  is  manifestly  present  in  certain  albumoses.  On 
hydrolvtic  decomposition  with  mineral  acids,  on  the  other  hand, 
giycocoU  can  be  obtained  from  most  albumins,  but  is  especially 
abundant  in  collagen,  viz.,  gelatin.  Two  exceptions  to  this  general 
rule,  however,  are  noted,  viz.,  casein^  and  (according  to  Magnus- 
Levy)  the  peculiar  albuminous  substance  which  is  known  as  the 
Bence  Jones'  body,  and  from  either  of  these  it  is  also  impossible  to 
obtain  a  hetero-albumose.  The  hetero-albumose  of  fibrin,  according^ 
to  Spiro,  yields  a  considerable  amount  of  glycocoll,  while  from  the 
proto-albumose  it  cannot  be  obtained. 

Heretofore  the  isolation  of  glycocoll  and  its  recognition  as  such 
were  attended  with  great  difficulties.  A  somewhat  simpler  pro- 
cedure, however,  has  recently  been  suggested  by  Baum,  and  with 
its  aid  Spiro  was  able  to  show  that,  contrary  to  former  views,  the 
substance  can  be  obtained  not  only  from  the  albuminoids,  but  also 
from  the  native  albumins,  with  the  exceptions  indicated.  The 
method  is  based  upon  the  observation  that  glycocoll  can  be  trans- 
formed into  hippuric  acid  in  the  test-tube  by  treating  with  benzoyl 
chloride  in  the  presence  of  sodium  hydrate,  and  that  the  formation 
of  the  resulting  hippuric  acid  can  be  readily  demonstrated  by  con- 
densing this  with  benzaldehyde  in  the  presence  of  sodium  acetate 
and  acetic  anhydride.  The  lactimide  of  benzoyl-amido-cinnamic 
acid  is  thus  formed.  On  decomposition  with  sodium  hydrate  this 
yields  phenyl-pyro-racemic  acid,  which  in  ethereal  solution  gives  a 

»  K,  Fischer  states  that  casein  contains  traces  of  glycocoll. 


THE  END-PRODUCTS  OF  ALBUMINOUS  DIGESTION.       211 

green  color  on  treating  with  chloride  of  iron.  With  phenyl- 
hydrazin,  moreover,  it  forms  an  osazon  which  melts  at  161°  C. 
These  changes  may  be  represented  by  the  equations  : 

(1)  CH2.(NH2).COOH  +  CeHs.COCl  =  CHj.NH(C6H5.CO).COOH  +  HCl 
GlycocoU.  Benzoyl  chloride.  Hippuric  acid. 

C2)  CH2.NH(CfiH5.CO).COOH  +  CgHsCOH  =  CgHs.CO.N.CrCH.CeHj  +  2H,0 

Hippuric  acid.  Benzaldehyde.  I  / 

CO 

Lactimide. 

(3)  CeHa.CO.N.CiCH.CeHs  CeHj.CONH^— C— COOH 

1/  +  H,0  =  ,  II 

CO  CH-CgHs 

Lactimide.  Benzyol-amido-cinnamic  acid. 

(4)  CeHs.CONH^— C— COOH 

II  +  H2O  =  CfiHgCO.NH^  +  CgHs.CHj-CO.COOH 
CH.  CgH-  Benzamide.  Phenyl-pyro-racemic 

Benzyol-amido-cinnamic  acid. 

acid.  ■( 

Method. — The  decomposition  of  the  albumins  (gelatin)  is  effected 
by  prolonged  boiling  with  dilute  sulphuric  acid — 25  per  cent,  solution. 
The  excess  of  acid  is  removed  with  plumbic  carbonate.  The  filtrate 
is  concentrated,  freed  from  any  tyrosin  that  may  have  separated  out, 
and  then  benzoylated  with  benzoyl  chloride  in  the  presence  of 
sodium  hydrate.  Care  should  be  had  that  the  reaction  of  the  solu- 
tion is  constantly  alkaline  during  this  process.  The  hippuric  acid 
is  then  extracted  with  acetic  ether.  The  dried  substance  is  now 
treated  with  tliree  molecules  of  acetic  anhydride,  one  molecule  of 
sodium  acetate,  and  one  molecule  of  benzaldehyde.  The  mixture  is 
heated  on  a  water-bath  for  half  an  hour.  The  condensation-produce 
is  then  treated  with  water  and  gently  warmed.  The  oil  that  sepa- 
rates out  is  dissolved  in  hot  alcohol  and  allowed  to  cool.  The  lacti- 
mide then  crystallizes  out,  and  can  be  recognized  as  follows:  the 
substance  is  heated  with  a  strong  solution  of  sodium  hydrate 
until  a  distinct  odor  of  ammonia  is  noticed.  This  is  due  to 
the  decomposition  of  the  benzamide.  On  acidifying  the  solution 
the  phenyl-pyro-racemic  acid  separates  out  and  can  be  readily  ex- 
tracted by  shaking  with  ether.  One  portion  of  the  ethereal  extract 
is  treated  with  a  dilute  solution  of  the  sesquichloride  of  iron,  when 
on  agitation  the  watery  layer  assumes  a  dark-green  color,  which 
gradually  changes  to  a  characteristic  yellow.  The  other  portion  is 
treated  with  an  ethereal  solution  of  phenylhydrazin,  which  leads  to  the 
separation  of  the  hydrazon  of  phenyl-pyro-racemic  acid.  After  wash- 
ing with  ether  this  may  be  identified  by  its  melting-point — 161°  C. 

As  regards  the  general  properties  of  glycocoll  and  its  preparation 
as  such,  see  pages  87  and  278). 

Tryptophan. — Tliis  substance  is  apparently  always  formed  when 
the  tryptic  digestion  of  the  albumins  has  extended  beyond  the  forma- 
tion of  albumoses.  As  its  presence  among  the  various  digestive 
products  is  easily  recognized,  it  is  thus  possible  to  ascertain  whether 


212  THE  PRODUCTS  OF  ALBUMINOUS  DIGESTION. 

the  destruction  of  the  albuminous  molecule  has  extended  to  the 
formation  of  amido-acids,  Avithout  testing;  for  these  directly.  Like 
the  amido-acids,  it  is  also  formed  during  the  hydrolytic  decomposi- 
tion of  the  albumins  with  baryta-water,  and  likcM'ise  results  during 
the  process  of  intestinal  pntrefaction.  Of  special  interest  is  the  fact 
that  while  the  primary  albumoses  of  fibrin,  as  also  the  secondary 
albumose-A,  on  further  digestion  with  tr^^psin,  give  rise  to  the 
formation  of  tryptophan,  the  secondary  albumose-B  apparently  does 
not  contain  the  chromogenic  group. 

Hopkins  and  Cole  have  shown  that  tryptophan  is  skatol  amido- 
acetic  acid : 

/C(CH3)^ 
CbHZ           '>C.CH(NH2).C00H 
\I?H' 

To  the  presence  of  this  complex  in  the  albuminous  molecule  the 
reaction  of  Adamkiewicz  is  due  (see  page  38). 

With  chlorine  and  bromine  it  yields  at  least  three  colored  products, 
the  so-called  proteinochromes.  Of  the  bromine  products,  one  is  a 
bluish-violet  substance,  and  contains  about  35  per  cent,  of  bromine ; 
the  second  is  a  red  body,  with  27  per  cent. ;  and  the  third  a  brovrn 
pigment,  with  the  same  amount  of  bromine. 

According  to  Xencki,  a  certain  similarity  exists  in  the  percentage 
composition  of  the  red  pigment  with  hsematoporphyrin,  viz.,  l)iHru- 
bin,  and  of  the  brown  pigment  with  the  so-called  melanins.  Tryp- 
tophan, moreover,  like  hsematin  and  hsematoporphyrin,  yields  pyrrol, 
hydrogen  sulphide,  methyl  mercaptan,  indol,  and  skatol  on  fusion 
with  caustic  alkali. 

Test. — The  test  for  tryptophan  and  the  isolation  of  the  three 
known  pigments  is  conducted  as  follows  :  the  digestive  mixture 
is  acidified  with  acetic  acid  and  treated  with  two  and  one-half  times 
its  volume  of  saturated  bromine-water.  A  beautiful  reddish-violet 
precipitate  is  thus  formed,  which  increases  on  standing.  After 
twentv-four  hours  this  is  filtered  off.  On  the  further  addition  of 
bromine-water  the  brown  pigment  separates  out  on  standing.  The 
red  pigment  will  be  found  in  the  violet  precipitate,  and  can  be  iso- 
lated as  follows  :  the  precipitate  is  first  washed  with  water  and  then 
extracted  with  dilute  ammonia ;  this  extract  is  precipitated  with 
acetic  acid.  The  precipitate  is  separated  from  the  brown  filtrate, 
redissolved  in  very  dilute  ammonia,  again  precipitated  with  acetic 
acid,  and  washed  with  water.  It  is  then  extracted  with  amyl 
alcohol ;  this  dissolves  the  red  bodv.  The  alcohol  is  evaporated 
off  at  40°  C,  the  residue  dried  at  106°  C,  and  finally  washed  with 
ether.  The  violet  pigment  is  obtained  on  further  extraction  of  the 
violet  precipitate  with  a  little  stronger  solution  of  ammonia  than  in 
the  first  instance.  The  substance  is  precipitated  with  acetic  acid, 
well  washed  with  water,  and  extracted  with  95  per  cent,  alcohol. 
The  alcoholic  extract  is  evaporated  to  dryness  at  40°  C,  the  residue 
dried  at  106°  C.  and  washed  with  petroleum  ether. 


THE  END-PRODUCTS  OF  ALBUMINOUS  DIGESTION.      213 

To  isolate  the  brown  pigment,  finally,  the  second  bromine  pre- 
cipitate is  filtered  off,  washed  with  water,  dissolved  in  very  dilute 
ammonia,  reprecipitated  with  acetic  acid  and  washed  with  Avater, 
and  briefly  with  95  per  cent,  alcohol,  both  of  which  dissolve  a  por- 
tion of  the  pigment.  It  is  then  dried  and  washed  with  ether.  The 
resulting  product  is  almost  black. 


CHAPTER    X. 

BACTEEIAL  ACTION  IN  THE  INTESTINAL  TRACT. 

I  HAVE  pointed  out  in  a  preceding  chapter  that  the  gastric  juice 
possesses  marked  germicidal  and  antiseptic  properties,  so  that  a  large 
number  of  bacteria  which  are  constantly  swallowed  with  the  saliva 
and  the  food  are  subsequently  destroyed  in  the  stomach.  A  perfect 
barrier  to  the  invasion  of  micro-organisms,  however,  does  not  exist, 
and  after  having  passed  the  pylorus  they  are  placed  in  surround- 
ings which  are  in  all  respects  most  favorable  to  their  develop- 
ment. Here  they  take  an  active  part  in  the  decomposition  of  the 
various  food-stuffs  which  have  escaped  digestion  in  the  stomach,  and 
further  modify  the  digestive  products  which  have  already  been 
formed,  as  also  those  which  result  from  the  action  of  the  various 
intestinal  ferments.  The  greater  portion  of  the  products  of  normal 
digestion,  however,  escapes  the  specific  activity  of  the  bacteria,  and 
is  absorbed  in  a  form  which  can  be  utilized  by  the  body  for  purposes 
of  nutrition.  Formerly  it  was  supposed  that  the  biliary  acids  played 
an  important  part  in  preventing  undue  activity  on  the  part  of  the 
bacteria,  but  this  view  has  now  been  largely  abandoned,  and  we  are 
totally  ignorant  as  to  the  manner  in  which  the  body  here  protects 
itself  against  excessive  bacterial  action.  It  has  been  argued  that  an 
accumulation  of  the  decomposition-products  which  result  from  the 
action  of  bacteria  upon  the  various  food-stuffs  in  itself  inhibits  the 
further  activity  of  the  organisms,  but  Ave  can  hardly  regard  such  an 
explanation  as  valid  in  view  of  the  fact  that  in  the  intestiues  these 
decomposition-products  are  to  a  large  extent  absorbed,  and  it  seems 
more  probable  that  a  vital  activity  of  the  epithelial  cells  is  here 
of  prime  importance.  In  the  small  intestine  at  least,  where  peri- 
stalsis is  extremely  active,  and  where  the  intestinal  contents  are 
churned  in  such  a  manner  that  the  individual  particles  are  almost 
constantly  in  contact  with  the  intestinal  walls,  we  accordingly  find 
that  bacterial  action  is  not  nearly  so  extensive  as  in  the  large  intes- 
tine, where  the  opposite  conditions  prevail.  In  the  clinical  labo- 
ratory we  find,  as  a  matter  of  fact,  that  the  degree  of  intestinal 
putrefaction  increases  at  once  when  the  peristalsis  of  the  small 
intestine  is  impeded,  and  reaches  its  greatest  height  if  the  secretion 
of  hydrochloric  acid  becomes  arrested  at  the  same  time. 

In  former  years  a  tendency  existed  among  physiologists  to  regard 
bacterial  action  in  the  intestine  as  serving  a  useful  ]iurposc,  and  it 
was  even  supposed  that,  as  in  the  case  of  plants,  animal  life  could 
not  go  on  in  the  absence  of  micro-organisms  from  the  alimentary 

214 


BACTERIAL  ACTION  IN  THE  INTESTINAL  TRACT        215 

canal.  This  view  has  now  been  abandoned,  however,  especially 
since  Thierfelder  and  Nuttall  were  able  to  demonstrate  that  guinea- 
pigs,  after  removal  from  the  uterus  of  the  mother  by  Csesarean 
section,  can  be  maintained  in  perfect  condition  as  to  health  and 
body-weight  when  fed  on  sterile  food  and  when  furnished  with 
sterile  air  exclusively.  On  subsequent  examination  it  was  shown 
that  the  intestinal  contents  of  these  animals  were  also  sterile.  We 
may  thus  conclude  that  the  presence  of  bacteria  in  the  intestinal 
contents  is  at  best  unnecessary,  and  it  is  doubtful,  indeed,  whether 
they  serve  a  useful  purpose  at  any  time. 

The  action  of  bacteria  upon  the  food-stuifs  is  in  certain  respects 
quite  analogous  to  that  of  the  digestive  ferments  which  are  fur- 
nished by  the  digestive  glands  of  the  animal  body.  The  primary 
digestion  of  the  original  material,  however,  does  not  cease  with  the 
production  of  substances  which  the  animal  can  subsequently  utilize 
for  the  purpose  of  replacing  tissue,  but  is,  on  the  whole,  far  more 
extensive.  Polysaccharides  and  disaccharides  are  thus  not  only 
inverted  to  monosaccharides,  but  the  latter  are  subsequently  further 
decomposed  into  material  in  which  but  little  potential  energy,  if 
any,  remains  stored.  Albumins  are  similarly  decomposed,  with 
the  ultimate  formation  of  substances  which  in  part  at  least  are  dis- 
tinctly toxic ;  and  the  fats  are  divided  into  their  components,  which 
are  then  further  broken  down,  with  the  final  formation  of  fatty 
acids  of  the  lowest  order,  etc.  A  great  variety  of  decomposition- 
products  thus  result  from  the  normal  food-stuffs,  which  are  further 
increased  by  those  arising  from  material  which  the  ferments  of 
the  animal  itself  are  incapable  of  digesting.  To  these  are  added 
the  decomposition-products  of  the  various  biliary  constituents  and 
of  the  albuminous  secretions  which  are  poured  into  the  intestinal 
canal  by  the  digestive  glands  themselves. 

As  has  been  pointed  out,  the  most  intense  degree  of  bacterial 
action  is  observed  in  the  large  intestine,  and  it  is  interesting  to 
note  that  while  albuminous  putrefaction  here  prevails,  the  fermenta- 
tive processes  in  the  more  restricted  sense  of  the  term,  viz.,  the 
decomposition  of  carbohydrates  and  fats,  occur  almost  exclusively  in 
the  small  intestine.  This  difference  may  be  dependent  to  a  certain 
degree  upon  the  difference  in  the  reaction  of  the  intestinal  contents 
in  the  two  sections  of  the  gut — that  of  the  small  intestine  in  its  lower 
portion  at  least  being  acid,  while  the  reaction  of  the  contents  of  the 
large  intestine  is  usually  alkaline.  But  it  is  also  possible  that  other 
and  still  unknown  factors  determine  this  difference,  and  that  the 
varying  reaction  is  primarily  due  to  the  decomposition-products 
directly  which  result  from  the  action  of  the  bacteria.  Among  these 
factors  the  relative  amount  of  water  may  be  of  importance. 

Nencki,  MacFadyen,  and  Sieber,  who  had  occasion  to  study  the 
chemical  composition  of  the  intestinal  contents  in  a  patient  in  whom 
an  artificial  anus  had  been  established  at  the  distal  end  of  the  ileum, 
give  the  following  account  of  their  observations  :     The  reaction  was 


216        BACTERIAL  ACTION  IN  THE  INTESTINAL   TRACT. 

quite  constantly  acid,  owing  to  the  presence  of  organic  acids,  and 
notably  of  acetic  acid.  Other  acids  that  were  present  were  lactic 
acid,  paralactic  acid,  various  volatile  fatty  acids,  succinic  acid,  and 
the  biliary  acids.  The  odor  but  rarely  suggested  the  existence  of 
putrefactive  changes.  Indol,  skatol,  and  phenol  could  not  be 
demonstrated  as  such,  although  the  urine  contained  indiean  on 
several  occasions.  Leucin  and  tyrosin  were  not  found.  Alcohol 
could  always  be  demonstrated.  Of  gases,  carbon  dioxide  Avas  ob- 
served, as  also  faint  traces  of  hydrogen  sulphide,  while  methyl- 
mercaptan  was  absent. 

Carbohydrate  fermentation  thus  manifestly  stands  in  the  fore- 
ground, and  is  exemplified  in  various  types  by  the  equations  : 

(1)  CgHjjOg  =  2C2H5.OH  +  2CO2,  alcoholic  fermentation. 

(2)  C2H5.OH  +  20  =  CH3.COOH  4-  H2O,   acetic  acid  fermentation. 

(3)  C6H,206  =  2CH3.CH(OH)COOH,  lactic  acid  fermentation. 

(4)  2C3H6O3  =  C3H..COOH  +  2CO2  +  4H,  butyric  acid  fermentation. 

The  products  of  albuminous  putrefaction,  on  the  other  hand,  are 
almost  exclusively  formed  in  the  large  intestine.  Primarily  they 
are  in  part  at  least  the  same  as  those  which  result  from  the  action 
of  trypsin  on  albumins,  and  in  experiments  in  vitro  we  thus  find 
albumoses,  peptone-like  bodies,  tryptophan,  leucin,  tyrosin,  aspara- 
ginic acid,  and  glutaminic  acid.  In  the  contents  of  the  large  intes- 
tine, however,  these  substances  are  found  only  in  traces,  so  that  we 
are  forced  to  the  conclusion  that  they  are  either  absorbed  as  soon  as 
formed  or  that  they  are  further  decomposed.  Both,  no  doubt, 
occurs,  and  related  bodies  are,  as  a  matter  of  fact,  encountered  in 
the  feces.  As  a  result  of  bacterial  activity  still  other  substances 
are  formed,  however,  which  are  apparently  not  derived  from  the 
final  products  of  digestion,  but  which  are  formed  from  the  more  or 
less  intact  albuminous  molecule  directly. 

The  more  important  decomposition-products  which  result  from 
the  action  of  bacteria  upon  the  products  of  albuminous  digestion  are 
here  considered. 

Indol. — Indol  is  a  derivative  of  the  tryptophan  complex,  viz.,  of 
skatol-amido-acetic  acid  : 


NH' 


CgH/  ^C.CH(NH2).C00H 

\-« —   — 


Structurally  it  is  closely  related  to  indigo,  and  according  to 
Nencki  this  transformation  can  be  effected  through  the  action  of 
ozone.     It  is  represented  by  the  equation  : 

/CH  ^  /  CO  V  /  CO  . 

2C6H  /        >CH    +   40   =    CeH  /         >C  =  C(         ^CeH,  +   2H2O. 

Indol.  Indigo. 

Conversely,  indigo  can  be  transformed  into  indol  on  reduction. 


SKATOL.  217 

From  the  albumins  the  substance  can  also  be  obtained  on  fusion 
with  potassium  hydroxide  (see  page  42). 

The  greater  portion  of  the  indol  that  is  formed  in  the  large  in- 
testine is  no  doubt  eliminated  in  the  feces.  A  certain  amount, 
however,  is  absorbed,  and  after  oxidation  to  indoxyl  appears  in  the 
urine  in  combination  with  sulpiiuric  acid  as  so-called  indican 
(pages  90  and  269).  If  larger  quantities  are  formed,  a  variable 
fraction  is  further  eliminated  in  the  urine  as  an  indoxyl  compound 
of  glucuronic  acid. 

Indol  crystallizes  in  small  platelets,  which  melt  at  52°  C,  and 
are  soluble  in  hot  water,  ether,  alcohol,  and  benzol.  Its  odor  is 
feculent ;  it  is  quite  volatile,  and  when  boiled  Avith  water  passes  over 
into  the  distillate.  With  picric  acid  it  forms  a  beautifully  red  crys- 
talline compound,  which  is  readily  decomposed,  however,  on  boil- 
ing with  dilute  ammonia,  and  the  liberated  indol  is  then  found  in 
the  distillate.  On  distilling  in  the  presence  of  sodium  hydrate,  on 
the  other  hand,  the  indol  is  decomposed. 

Tests. — When  treated  in  aqueous  solution  with  nitric  acid  and  a 
trace  of  sodium  nitrite  a  red  precipitate  of  the  nitrate  of  nitroso- 
indol  is  formed.  This  is  soluble  in  alcohol  and  crystallizes  out  upon 
the  addition  of  ether. 

If  a  small  piece  of  pine  wood  is  moistened  with  strong  hydro- 
chloric acid  and  then  placed  in  a  watery  solution  of  indol,  it  gradu- 
ally assumes  a  cherry-red  color. 

An  aqueous  solution  of  indol  is  treated  with  a  small  amount  of 
a  solution  of  sodium  nitroprusside  until  a  brownish-yellow  color 
develops.  If  now  a  dilute  solution  of  sodium  hydrate  is  added 
drop  by  drop,  the  color  changes  to  violet.  Upon  the  further  addi- 
tion of  a  little  dilute  hydrochloric  acid  this  becomes  a  deep  blue, 
while  an  excess  of  the  acid  destroys  the  blue  color. 

For  the  isolation  of  indol,  see  page  224). 

Skatol. — Skatol,  like  indol,  is  a  direct  derivative  of  tryptophan, 
and  is  likewise  formed  during  the  process  of  albuminous  putrefac- 
tion. It  is  a  methylated  indol,  and  may  be  represented  by  the 
formula : 

yC(CH3)^ 

By  combining  with  carbon  dioxide  it  gives  rise  to  the  formation 
of  skatol-carbonic  acid,  which  is  also  found  in  the  contents  of  the 
large  intestine,  and  belongs  to  the  ortho-series.     Its  formula  is 

CfiHZ  "^C.COOH. 

Like  indol,  skatol  is  also  formed  on  fusing  albumins  with  caustic 
soda,  and  can  be  obtained  from  indigo  on  reduction  with  tin  and 
hydrochloric  acid.     When  passed  through   a  red-hot  tube  it  yields 


218        BACTERIAL  ACTION  IN  THE  INTESTINAL   TRACT. 

indol.     On  absorption,  it  is  oxidized  to  skatoxyl  and  is  eliminated 
in  the   urine  in  combination  Avith  sulphuric  acid  and   glucuronic 
acid,  as  in  the  case  of  indol  (see  pages  90  and  272).     Skatol-car- 
bonic  acid,  on  the  other  hand,  appears  in  the  urine  as  such. 
Another  derivative  of  skatol  is  Bauni's  skatosin — CioH„3X,02. 

Skatol  crystallizes  in  fine  platelets,  which  melt  at  95°  C.  aiid  are 
readily  soluble  in  ether,  alcohol,  and  benzol ;  in  hot  water  it  is 
soluble  with  greater  difficulty  than  indol.  Its  odor  is  exceedingly 
oiiensive.  Like  indol,  it  is  volatile,  and  combines  with  picric  acid 
to  form  a  red  crystalline  compound.  On  distilling  this  in  ammo- 
niacal  solution  or  in  the  presence  of  sodium  hydrate  the  skatol  passes 
over  as  such,  while  indol  in  the  latter  instance  is  decomposed.  On 
distilling  a  mixture  of  indol  and  skatol  in  aqueous  solution  the 
skatol  i)asses  over  first,  and  it  is  thus  possible  to  separate  the  two 
substances  from  each  other. 

Tests. — From  its  aqueous  solutions  skatol  is  precipitated  by  yellow 
nitric  acid  as  a  white  substance — skatol  nitrate. 

If  a  small  piece  of  pine  wood  is  moistened  with  an  alcoholic  solu- 
tion of  skatol  and  then  placed  in  strong  hydrochloric  acid,  it  assumes 
a  red  color.  If,  on  the  other  hand,  the  test  is  conducted  as  with 
indol,  no  reaction  is  obtained. 

AVith  nitric  acid  of  a  specific  gravity  of  1.2  skatol  gives  a  marked 
xanthoproteic  reaction  on  boiling — i.e.,  a  yellow  color  which  changes 
to  orange  when  ammonia  is  added  in  excess. 

The  substance  does  not  give  the  reaction  with  sodium  nitro- 
prusside. 

Isolation  (see  page  224). 

Phenol. — The  phenol  which  is  formed  during  the  process  of 
intestinal  putrefaction  is  derived  from  tyrosin.  As  has  been  shown, 
this  is  first  reduced  to  hydroparacumaric  acid.  This  in  turn  is 
oxidized  to  para-oxy-phenyl-acetic  acid.  Paracresol  then  is  formed 
through  a  splitting  ofF  of  carbon  dioxide,  and  on  subsequent  oxida- 
tion phenol  results  (see  page  89).  To  a  certain  extent  this  is  elimi- 
nated in  the  feces,  but  a  variable  amount  is  always  absorbed,  and 
subsequently  oxidized  in  part  to  hydroquinon  or  pyrocatechin. 
These  three  bodies  then  combine  with  sulphuric  acid  and  are  elimi- 
nated through  the  urine  in  this  form.  A  certain  amount  of  para- 
cresol, moreover,  is  absorbed  as  such,  and  likewise  appears  in  the 
urine  as  a  conjugate  sulphate.  According  to  some  observers,  indeed, 
a  larger  quantity  of  paracresol  is  here  encountered  than  of  phenol. 

Tests. — An  aqueous  solution  of  phenol  when  treated  with  a  few 
drops  of  a  solution  of  the  sesquichloride  of  iron  assumes  an  amethyst 
color,  which  becomes  especially  apparent  on  further  dilution  with 
water  if  much  phenol  is  present. 

With  bromine-water  a  crystalline  precipitate  of  tribromophenol  is 
obtained. 

With  Millon's  reagent  a  red  color  develops,  which,  however,  is 
common  to  other  bodies  of  this  series  as  well  (see  page  34). 

Isolation  (see  page  224). 


BACTERIAL  DECOMPOSITION  OF  THE  FATS.  219 

In  addition  to  phenol,  indol,  skatol,  and  skatol-carbonic  acid,  as 
also  the  two  hydroxylated  benzol-derivatives  of  tyrosin,  viz.,  para- 
oxy-phenyl-propionic  acid  (hydroparacumaric  acid)  and  para-oxy- 
phenyl-acetic  acid,  we  further  meet  with  two  non-hydroxylated 
aromatic  acids,  which  are  homologous  with  benzoic  acid,  viz.,  phenyl- 
propionic  or  hydrocinnamic  acid  and  phenyl-acetic  acid.  Accord- 
ing to  Salkowski,  these  may  develop  directly  from  the  albuminous 
molecule,  but  may  also  result  from  tyrosin  (see  page  95). 

The  non-nitrogenous  aromatic  acids  are  in  part  eliminated  in  the 
feces.  To  some  extent,  however,  they  are  also  absorbed.  The 
hydroxylated  acids  are  then  eliminated  in  the  urine  either  as  such, 
or,  like  phenol,  indol,  and  skatoxyl,  in  combination  with  sulphuric 
acid,  while  the  non-hydroxylated  acids  combine  with  glycocoll,  and 
are  eliminated  as  hippuric  acid  and  phenaceturic  acid,  as  already 
described  (page  95). 

As  regards  the  fate  of  the  small  amounts  of  leucin,  asparaginic 
acid,  and  glutaminic  acid  which  are  also  formed  during  the  process 
of  albuminous  putrefaction,  it  seems  that  they  are  usually  absorbed, 
and  are  then  further  decomposed  within  the  body  of  the  animal.  To 
a  slight  extent,  however,  this  decomposition  also  takes  place  within 
the  large  intestine,  and  we  accordingly  meet  with  small  amounts 
of  succinic  acid,  glutaric  acid,  capronic  acid,  valerianic  acid,  butyric 
acid,  and  acetic  acid.  The  sulphur  of  the  albuminous  molecule  is 
usually  set  free  in  the  form  of  hydrogen  sulphide,  but  traces  of 
methyl-mercaptan  are  also  frequently  observed,  and  still  further 
contribute  to  the  offensive  odor  of  the  feces.  Whether  these  sulphur 
bodies  result  from  decomposition  of  the  tryptophan,  is  not  known. 

Of  the  gases  which  are  constantly  present  in  the  contents  of  the 
large  intestine,  methane  further  deserves  especial  mention.  It  is  to 
a  great  extent,  no  doubt,  referable  to  the  peculiar  form  of  fermenta- 
tion to  which  the  celluloses  are  subject.  But  in  part  at  least  it  prob- 
ably also  results  from  the  decomposition  of  the  fatty  acids  and  of 
cholin. 

Ptomains  are  normally  not  found  in  the  intestinal  contents,  but 
may  be  encountered  under  certain  pathological  conditions.  In  Asiatic 
cholera  and  in  cases  of  cystinuria  putrescin  and  cadaverin  have  thus 
been  isolated,  and  in  other  diseases,  no  doubt,  they  also  occur. 

The  methods  which  are  employed  for  the  purpose  of  isolating  the 
more  important  products  of  albuminous  putrefaction  are  described 
in  the  chapter  on  the  Feces. 

BACTERIAL  DECOMPOSITION  OF  THE  FATS. 

As  in  the  case  of  the  carbohydrates  and  albumins,  a  comparatively 
small  portion  of  the  fats  only  undergoes  bacterial  decomposition,  and 
it  appears  that  this  principally  occurs  in  the  lower  portion  of  the 
small  intestine.  As  in  the  case  of  the  steapsin  of  the  pancreatic 
juice,  the  neutral  fats  are  thus   first  decomposed  into  glycerin  and 


220        BACTERIAL  ACTION  IN  THE  INTESTINAL   TRACT. 

the  correspondint^  f\ittv  acids,  but  the  process  extends  furthur 
and  as  a  result  a  gradual  reduction  of  the  higher  acids  to  the  lowest 
forms  takes  place.  To  a  certain  extent  these  are  then  absorbed  and 
further  decomposed  in  the  body,  but  a  not  inconsiderable  portion  is 
directly  eliminated  in  the  feces,  and  we  accordingly  find  here  repre- 
sentatives of  the  group,  from  palmitic,  stearic,  and  oleic  acids  down 
to  butyric  acid  and  acetic  acid.  The  glycerin  is  absorbed,  and  is  to 
a  certain  extent  no  doubt  utilized  by  the  epithelial  cells  in  the  syn- 
thesis of  fats. 

The  lecithins  are  decomposed  in  the  same  manner  as  under  the 
influence  of  steapsin,  with  the  formation  of  glycerin-phosphoric  acid, 
fatty  acids,  and  cholin.  Whether  or  not  the  latter  may  then  be 
transformed  into  neurin  is  not  known,  but  under  normal  condi- 
tions this  probably  does  not  occur.  The  glycerin-phosphoric  acid 
is  subsequently  no  doubt  absorbed  together  with  some  of  the  fatty 
acids,  ajid  appears  in  the  urine  as  such.  The  cholin,  on  the  other 
hand,  is  further  decomposed,  with  the  formation  of  ammonia,  carbon 
dioxide,  and  methane. 

BACTERIAL   DECOMPOSITION    OF    THE    BILIARY   CON- 
STITUENTS. 

In  former  years  it  was  generally  supposed  that  the  biliary  acids 
after  their  elimination  into  the  intestinal  canal  were  there  largely 
absorbed  and  returned  to  the  liver,  while  a  smaller  portion  was 
decomposed  and  eliminated  in  the  feces. 

Within  more  recent  years  there  has  been  a  tendency  among 
physiologists  to  deny  the  existence  of  a  circulation  of  the  bile  acids, 
on  the  basis  principally  that  bile  acids  could  normally  not  be 
demonstrated  in  the  blood  or  in  the  urine.  Croftan,  however, 
has  shown  that  Rafter  all  such  a  circulation  may  exist.  He  suc- 
ceeded in  demonstrating  the  presence  of  biliary  acids  in  the  blood- 
cells,  and  further  showed  that  they  are  without  doubt  present  in 
the  leucocytes.  Occurring  in  this  form  it  suggests  itself  that  they 
play  the  role  of  foreign  matter  and  do  not  enter  into  account 
physiologically. 

That  portion  of  the  bile  acids  which  is  not  resorbed  is  decomposed 
in  the  intestinal  canal,  and  in  the  human  being  dyslysins  only  are 
encountered  in  the  feces  while  the  amido  radicles  have  apparently 
been  further  broken  down.  In  other  animals  glycocholic  acid  has 
been  found,  but  taurocholic  acid  apparently  always  succumbs  to  the 
action  of  the  bacteria.  Of  the  fate  of  the  amido-radicles  we  know 
little,  but  it  is  possible  that  both  are  in  part  further  decomposed  and 
in  part  absorbed.  Taurin  may  then  appear  in  the  urine  either  as 
such  or  as  tauro-carbaminic  acid  ;  but  it  may,  on  the  other  hand,  again 
combine  with  cholalic  acid  and  reappear  in  the  bile.  -  The  glycocoll 
similarly  may  in  part  be  transformed  into  urea ;  or  it  may  combine 
with  the  non-hydroxylated  aromatic   acids  which  are  also  formed 


DECOMPOSITION  OF  THE  BILIARY  CONSTITUENTS.      221 

during  the  process  of  intestinal  putrefaction,  and  appear  in  the 
urine  as  hippuric  acid  and  phenaceturic  acid ;  or  it  may  find  its  way 
to  the  liver  and  be  re-eliminated  into  the  intestinal  tract  as  glyco- 
cholic  acid. 

Bilirubin  is  reduced  to  hydrobilirubin  during  the  process  of 
intestinal  putrefaction  and  largely  eliminated  in  the  feces  as  such. 
This  reduction,  according  to  Nencki,  MacFadyen,  and  Sieber, 
occurs  in  man,  in  the  large  intestine.  A  portion,  however,  is  prob- 
ably absorbed  and  eliminated  in  the  urine  as  urobilin. 


CHAPTER    XI. 

THE  FECES. 

I  HAVE  shown  in  the  preceding  chapters  that  the  greater  portion 
of  the  ingested  food  is  transformed  in  the  gastro-intestinal  canal 
into  material  which  can  be  utilized  by  the  body  for  purposes  of 
nutrition,  and  is  there  absorbed.  A  certain  proportion,  however^ 
invariably  escapes  digestion,  and  is  partly  decomposed  by  the 
bacteria  of  the  intestinal  canal  into  the  various  substances  which 
have  been  considered  in  the  preceding  chapter.  These  substances 
in  turn  are  in  part  absorbed,  and  are  partly  eliminated  in  the  feces, 
together  with  particles  of  undigested  food  and  undigestible  material 
which  have  passed  through  the  digestive  tract  as  such.  In  addition 
we  find  here  the  various  native  and  decomposition-products  of  the 
bile,  the  pancreatic  juice,  the  enteric  juice,  in  so  far  as  they  have  not 
been  absorbed,  together  with  intestinal  mucus,  desquamated  epithe- 
lial cells,  and  bacteria. 

Consistence  and  Form. — The  consistence  and  form  of  the  feces 
are  principally  dependent  upon  the  amount  of  water  that  is  present, 
and  vary  in  different  animals.  Generally  speaking,  they  are  softer 
in  the  herbivorous  animals  than  in  the  carnivora.  In  man  they 
usually  occur  in  the  characteristic  plastic,  cylindrical  form,  but  they 
may  at  times  be  mushy,  or  round  and  hard,  even  in  health. 

Amount. — The  amount  of  fecal  material  which  is  eliminated  in 
the  twenty-four  hours  depends  primarily  U])on  the  amount  and  the 
character  of  the  food  that  has  been  ingested.  In  man  it  normally 
varies  between  100  and  200  grammes,  but  may  diminish  to  60 
grammes  or  rise  to  250  grammes,  even  in  health,  according  to  the 
preponderance  of  animal  food  or  of  vegetable  material,  which  has 
entered  into  the  composition  of  the  diet. 

Odor. — The  disagreeable  odor  of  the  feces  is  largely  due  to  indol 
and  skatol,  but  may  be  further  increased  by  the  presence  of  hydro- 
gen sulphide,  methane,  and  methyl-mercaptan. 

Color. — The  color  varies  with  the  character  of  the  food  ingested, 
and  is  usually  but  little  influenced  by  the  decomposition-products 
of  the  biliary  pigments.  In  carnivorous  animals  the  feces  are  almost 
black,  owing  to  the  presence  of  hajmatin  and  sulphide  of  iron.  In 
adult  man  the  color  normally  varies  from  a  light  to  a  dark  brown. 
In  infants  in  which  the  bile-pigments  appear  as  such  the  feces  are 
of  a  bright-yellow  or  a  greenish-yellow  color. 

At  times  and  apparently  under  normal  conditions  stools  are  also 
passed  which  are  grayish  white  in  color  and  closely  resemble  the 
so-called  acholic  stools  which  are  observed   in  cases  of  biliary  ob- 

222 


GENERAL  CHEMICAL  COMPOSITION.  223 

struction.  Fats,  however,  are  not  necessarily  present  in  increased 
amounts,  and  there  is  no  reason  to  assume  that  the  biliary  passages 
are  not  patent  or  that  no  bile  is  being  secreted.  Possibly  the  lack 
of  color  in  such  stools  is  referable  to  the  formation  of  colorless 
decomposition-products  of  bilirubin,  such  as  the  leuko-urobilin  of 
Nencki,  and  in  the  last  instance  to  the  presence  in  the  intestinal 
caual  of  micro-organisms  which  are  usually  absent.  Nothing  defi- 
nite is,  however,  as  yet  known  of  the  conditions  which  favor  the  for- 
mation of  such  products. 

Macroscopic  Constituents. — On  macroscopic  examination  of  the 
feces  we  frequently  find  undigested  particles  of  food,  such  as  skins 
of  berries,  large  pieces  of  connective  tissue,  woody  vegetable  fibres, 
undigested  pieces  of  apples,  pears,  potatoes,  grains  of  corn,  flakes  of 
casein,  etc. 

Microscopic  Constituents. — On  microscopic  examination  we 
usually  find  undigested  bits  of  muscle-fibre,  connective-tissue  of  the 
white  fibrous  variety,  fragments  of  the  framework  of  vegetable 
matter,  often  still  enclosing  cells  with  starch-granules,  flakes  of 
casein,  globules  of  fat,  fatty  acid  needles,  crystals  of  calcium  oxalate, 
neutral  calcium  phosphate,  ammonio-magnesium  phosphate,  calcium 
lactate  (these  are  seen  especially  in  children  on  a  milk  diet),  and 
more  rarely  of  calcium  carbonate,  calcium  sulphate,  and  cholesterin. 
So-called  Charcot-Leyden  crystals,  which  consist  of  the  phosphate 
of  spermin,  are  in  my  experience  only  found  under  pathological  con- 
ditions. We  further  meet  with  more  or  less  disintegrated  epithelial 
cells,  a  few  leucocvtes,  bits  of  mucus,  and,  above  all,  with  innumer- 
able micro-organisms.  Often,  indeed,  it  appears  as  though  the 
stools  consist  of  these  exclusively.  Their  number,  even  in  health, 
is  enormous.  SucksdorfF  thus  found  in  his  own  person  that  on  an 
average  53,124,000,000  were  eliminated  in    the  twenty-four  hours. 

Reaction. — In  adult  man  the  reaction  of  the  stools  is  usually 
alkaline,  sometimes  neutral,  and  but  rarely  acid.  Acid  stools,  on  the 
other  hand,  are  the  rule  in  infants. 

General  Chemical  Composition. — A  general  idea  of  the  average 
composition  of  the  human  feces  may  be  formed  from  the  folloA^-ing 
analyses,  which  are  taken  from  Gautier,  and  have  reference  to  1000 
parts  by  weight  of  the  fresh  material : 

Adult  man.  Suckling. 

Water      744.00  871.3 

Solids      267.00  148.7 

Total  organic  matter 208.75  137.1  ^ 

Total  mineral  matter 10.95*  13.6 

Alimentary  residue 84.00 

The  organic  material  yielded  : 

Aqueous  extract 53.40  53.5 

Alcoholic  extract      41.65  8.2 

Ethereal  extract 30.70  17.6  * 

'Includinsr  54  parts  of  mucus,  epithelium,  and  calcareous  salts. 

2  Not  comprising  earthy  phosphates. 

3  Of  this,  0.2  parts  of  cholesterin. 


224  THE  FECES. 

The  individual  constituents  of  the  feces  may  be  grouped  as 
follows  : 

1.  Food-material  which  has  escaped  the  process  of  digestion,  and 
of  bacterial  decomposition,  such  as  starches,  muscle-tissue,  connective- 
tissue,  fats,  etc. 

2.  Undigestible  material,  which  has  been  ingested  as  such,  or 
which  has  resulted  from  the  decomposition  of  complex  substances 
which  are  partly  digestible,  such  as  gums,  pectins,  resins,  chitin, 
chlorophyl,  hsematin,  and  insoluble  silicates,  sulphates,  phosphates, 
etc. 

3.  Derivatives  of  the  bile,  such  as  the  dyslysins,  cholesterin,  and 
exceptionally  the  native  biliary  acids  as  such,  and  further  hydrobili- 
rubin,  stercobilin,  etc. 

4.  Intestinal  mucus. 

5.  Products  of  albuminous  digestion,  such  as  albumoses,  peptone- 
like bodies,  leucin,  tyrosin,  asparaginic  acid,  and  glutaminic  acid. 

6.  Products  of  bacterial  action.  These  comprise  the  entire  series 
of  fatty  acids  from  acetic  acid  to  palmitic  acid ;  further,  lactic  acid, 
succinic  acid,  glutaric  acid,  leucin,  tyrosin,  hydroparacumaric  acid, 
para-oxv-phenyl-acetic  acid,  phenyl-propionic  acid,  phenyl-acetic 
acid,  phenol,  paracresol,  indol,  skatol,  skatol-carbonic  acid,  ammo- 
nium carbonate,  ammonium  sulphide,  and  conjugate  glucuronates. 

7.  Products  of  metabolism,  which  are  in  part  eliminated  through 
the  intestines,  such  as  uric  acid,  urea,  xanthin  bases,  etc. 

8.  Water. 

9.  Gases,  which  are  in  part  referable  to  the  various  fermentative 
and  putrefactive  processes  which  take  place  in  the  intestinal  canal, 
such  as  carbon  dioxide,  methane,  hydrogen,  hydrogen  sulphide, 
methyl-mercaptan,  and  phospliin.  The  nitrogen,  on  the  other  hand, 
Avhicii  is  also  constantly  met  with,  is  probably  derived  from  the 
blood,  and  has  in  part  been  swallowed. 

Many  of  these  substances  have  already  been  considered  in  detail, 
and  it  will  suffice  at  this  place  to  indicate  the  manner  in  which  the 
most  important  products  of  albuminous  putrefaction  can  be  isolated 
from  the  feces. 

ANALYSIS  OF  THE  PRODUCTS  OF  ALBUMINOUS 
PUTREFACTION. 

The  feces  are  diluted  Avith  water,  passed  through  a  muslin  filter 
to  remove  particles  of  food-material,  and  distilled  until  about  four- 
fifths  of  the  entire  volume  have  passed  over.  The  distillate  B  con- 
tains indol,  skatol,  phenol,  paracresol,  and  the  volatile  acids  which 
are  present  in  the  free  state,  w^hile  the  remaining  products  of  putre- 
faction are  found  in  the  residual  solution  A.  The  distillate  B  is 
neutralized  with  sodium  carbonate  and  redistilled.  This  second  dis- 
tillate, C,  contains  indol,  skatol,  phenol,  and  paracresol,  while  the 
volatile   acids  remain  behind   as  sodium   salts,   and   can  be  sepa- 


PRODUCTS  OF  ALBUMINOUS  PUTREFACTION.  225 

rated  from  each  other  according  to  the  usual  analytical  methods 
(see  page  284).  Distillate  C  is  now  rendered  alkaline  with  sodium 
hydrate  and  extracted  with  ether  by  shaking.  This  takes  up  the 
indol  and  skatol,  Avhich  may  be  obtained  in  crystalline  form  on 
evaporation  of  the  ether,  and  can  be  separated  from  each  other  by 
fractional  distillation  with  steam,  when  the  skatol  passes  into  the 
distillate  first.  The  residual  solution  of  C  contains  phenol  and 
paracresol  as  sodium  compounds.  This  is  now  acidified  with  sul- 
phuric acid  and  distilled,  when  the  phenols  pass  over,  and  may 
then  be  separated  from  each  other  as  described  elsewhere. 

The  residual  solution  A  is  now  concentrated  and  treated  with  a 
large  excess  of  alcohol,  in  order  to  precipitate  any  albumins  and 
mineral  salts  that  may  be  present.  The  alcoholic  filtrate  is  then 
transformed  into  an  aqueous  solution  and  acidified  with  sulphuric 
acid.  The  aromatic  acids  are  thus  set  free  and  are  extracted  with 
ether  by  shaking.  The  ethereal  solution  is  evaporated  to  dryness, 
the  residue  dissolved  in  a  small  amount  of  a  dilute  solution  of 
sodium  hydrate,  and  precipitated  with  barium  chloride.  The  fatty 
acids  are  thus  obtained  as  barium  soaps,  and  are  filtered  off.  They 
are  placed  in  water,  which  dissolves  the  salts  of  the  aromatic  acids. 
In  this  solution  the  acids  are  then  set  free  by  acidifying  with  sul- 
phuric acid,  and  are  extracted  with  ether.  The  ethereal  extract  is 
now  evaporated  to  dryness  and  the  free  acids  dissolved  in  water. 
On  distillation  in  a  current  of  steam,  phenyl-propionic  acid  and 
phenyl-acetic  acid  pass  over,  and  can  be  subsequently  separated 
from  each  other  by  fractional  distillation.  The  hydroxy lated  oxy- 
acids  and  skatol-carbonic  acid  remain  behind.  The  latter  separates 
out,  oh  further  concentration  of  the  solution  and  cooling,  in  the 
form  of  white  wart-like  granules,  while  the  oxy-acids  remain  behind, 
and  can  be  separated  from  each  other  by  means  of  their  varying 
solubility  in  benzol.  They  can  be  recognized  by  applying  Millon's 
test.  Skatol-carbonic  acid,  on  the  other  hand,  reacts  in  very  much 
the  same  manner  with  yellow  nitric  acid  as  does  indol,  but  the  red 
color  is  in  this  case  referable  to  a  different  pigment  (see  also  page 
273). 

Hydrobilirubin. — It  has  been  stated  that  bilirubin  under  the 
influence  of  bacterial  action  supposedly  undergoes  a  process  of  reduc- 
tion in  the  intestinal  canal  and  is  transformed  into  hydrobilirubin. 
It  is  assumed  that  as  such  it  is  then  in  part  absorbed  and  possibly 
appears  in  the  urine,  while  the  remaining  portion  is  eliminated  in 
the  feces.  According  to  some  observers,  it  is  identical  with  the 
stercobiUn  of  Vanlair  and  Masius.  The  spectrum  of  the  two  bodies 
is  very  similar,  but  while  solutions  of  hydrobilirubin  on  treatment 
with  chloride  of  zinc  and  ammonia  show  three  bands  of  absorption, 
stercobilin  is  said  to  give  rise  to  four  bands.  Garrod  claims  that 
stercobilin  is  identical  with  the  urobilin  of  the  urine,  and  differs  from 
hydrobilirubin  in  containing  a  much  smaller  percentage  of  nitrogen, 
viz.,  4.11,  as  compared  with  9.22.     According  to  the  same  observer, 

15 


226  THE  FECES. 

hydrobilirubiu  is  a  laboratory  product,  and  is  met  with  neither  in 
the  feces  nor  the  urine. 

Excretin. — This  is  a  substance  which  was  first  isolated  by 
Marcet  from  the  feces  of  herbivorous  animals,  but  is  said  to  occur 
also  in  human  stools.  According  to  Hinterberger,  it  has  the  formula 
CayHggO,  and  is  thus  closely  related  to  cholesterin. 

Stercorin. — Stercorin,  or  seroUn,  as  it  has  been  called,  is  a  sub- 
stance which  Flint  obtained  from  the  feces  of  man,  but  which  is 
probably  an  impure  form  of  cholesterin,  both  having  the  same 
general  reactions. 

MECONIUM. 

The  term  meconium  has  been  apj^lied  to  the  material  which  accu- 
mulates in  the  intestinal  tract  during  foetal  life,  and  which  is  expelled 
soon  after  birth.  Food-products  are  here,  of  course,  wanting,  and 
as  the  intestinal  tract  of  the  foetus  is  free  from  bacteria  the  meconium 
consists  essentially  of  mucus,  desquamated  epithelial  cells,  and  the 
normal  biliary  constituents  which  are  present  in  the  intestinal  tract 
before  birth.  We  accordingly  find  bilirubin  and  biliverdin,  the 
former  often  in  crystalline  form,  the  native  biliary  acids,  a  small 
amount  of  fatty  acids,  cholesterin,  and  mineral  salts,  while  hydro- 
bilirubin,  the  dyslysins,  leucin,  tyrosin,  indol,  skatol,  lactic  acid, 
albumoses,  etc.,  are  absent.  According  to  Zweifel,  it  contains  from 
79.8  to  80.5  per  cent,  of  water  and  from  19.5  to  20.2  per  cent,  of 
solids,  of  which  0.978  is  referable  to  mineral  ash,  0.797  to  choles- 
terin, and  0.772  to  fatty  acids. 

Its  color  is  a  dark  brownish-green,  and  the  reaction  usually  acid. 
In  general  appearance  it  resembles  pitch,  and  is  hence  also  spoken 
of  by  the  Germans  as  Kindspech  (infant  pitch). 


CHAPTER    XII. 

THE  URINE. 

The  urine  is  by  far  the  mdst  important  excretory  product  of  the 
animal  body,  and  the  medium  through  which  the  end-products  of 
nitrogenous  metabolism  and  soluble  mineral  salts  are  almost  exclu- 
sively eliminated  under  normal  conditions.  Abnormal  products  of 
metabolism  also,  and  many  substances  that  have  found  their  way 
into  the  circulation  from  without,  and  which  are  foreign  to  the  body, 
are  likewise  removed  in  this  manner,  either  as  such  or  in  a  more  or 
less  modified  form.  All  these  substances  are  found  in  the  urine  in 
aqueous  solution,  and  it  is  to  be  noted  that  of  the  total  amount  of 
water  which  is  daily  excreted  at  least  50  per  cent,  appears  in  this 
form. 

Formerly,  it  was  sujiposed  that  the  various  elements  which  are 
found  in  the  urine,  and  notably  the  mineral  salts  and  water,  were 
eliminated  by  a  simple  process  of  osmosis.  Later  it  was  siiown, 
however,  that  in  the  elimination  of  the  organic  constituents  at  least 
the  renal  epithelium  of  the  lunniferous  tubules  plays  an  active  part, 
and  it  now  appears,  indeed,  that  all  the  substances  which  occur  in 
the  urine,  including  a  certain  amount  of  water  even,  are  removed 
from  the  blood,  viz.,  the  lymph,  through  the  intervention  of  the 
epithelial  cells.  It  is  supposed,  moreover,  that  these  structures 
possess  certain  selective  properties,  and  we  can  accordingly  under- 
stand why  tlie  composition  of  the  blood  always  remains  constant. 

The  kidneys  cannot  be  regarded  as  simple  excretory  organs,  how- 
ever, for  we  know  that  im])ortant  synthetic  processes  also  take  place 
in  tliem,  the  object  of  which  is  to  transform  certain  substances  which 
may  occur  in  the  circulating  blood  into  compounds  that  can  be  more 
readily  eliminated. 

The  most  important  synthesis  of  this  kind  is  that  of  glycocoll  and 
benzoic  acid,  which  results  in  the  formation  of  hii)puric  acid  (see 
page  278). 

GENERAL  CHARACTERISTICS  OF  THE  URINE. 

The  general  appearance  of  the  urine  varies  in  different  animals. 
In  man  it  is  perfectly  transparent  when  recently  passed,  but  soon 
becomes  turbid,  and  on  standing  deposits  a  light,  flocculent  sediment, 
which  consists  of  a  mucinous  body  and  a  few  epithelial  cells  and 
leucocytes  that  are  derived  from  the  urinary  passages. 

In  addition,  a  small  number  of  crystals  of  uric  acid  or  of  oxalate 
of  calcium  may  also  be  seen.     The  supernatant  fluid  is  then  per- 

227 


228  THE   VRISE. 

fectly  clear,  and  remains  so  if  care  is  taken  to  prevent  the  access  of 
micro-ortranisms.  If  left  exposed  to  the  air,  however,  bacterial 
decomijo.-ition  soon  takes  place.  Ammonia  appears  in  the  free  state, 
and  as  a  conseqnence  of  the  change  in  reaction  certain  constituents 
of  the  urine  are  precipitated  and  render  the  liquid  turbid.  Sooner 
or  later  they  settle  to  the  bottom,  but  owing  to  the  presence  of 
innumerable  micro-organisms  the  supernatant  fluid  remains  cloudy. 
Such  urine  is  said  to  have  undergone  ammoniacal  decomposition. 

A  formation  of  sediments,  aside  from  the  light  cloud  which  de- 
velops in  every  urine  on  standing  for  a  short  while,  may,  however, 
also  occur  in  the  absence  of  micro-organisms.  In  the  winter-time  it 
is  a  common  experience  to  see  the  entire  volume  of  urine  become 
turbid  when  kej^t  in  a  cold  room.  This  is  owing  to  the  fact  that 
the  urates  of  the  urine  are  ver}'  much  less  soluble  in  cold  than 
in  warm  water,  and  are  hence  thrown  down.  On  standing,  they 
soon  settle  to  the  bottom,  and  the  supernatant  liquid  remains  clear 
so  long  as  bacterial  decomposition  does  n(jt  occur.  A  similar  forma- 
tion of  sediments  is  observed  if  the  reaction  of  the  urine  is  alkaline, 
owing  to  the  presence  of  fixed  alkali  in  contradistinction  to  free 
ammonia.  This  may  at  times  be  observed  after  a  large  meal,  or 
after  the  administration  of  sufficiently  large  amounts  of  alkalies  as 
such,  or  of  substances  which  are  oxidized  to  alkaline  carljonates 
within  the  body.  In  such  an  event  the  urine  may  be  clear  when 
first  passed,  but  after  standing  a  short  time  it  becomes  turbid, 
and  deposits  a  sediment  of  phosphates  and  carbonates  of  the  alka- 
line earths.  The  change  is,  no  doubt,  due  to  an  escape  of  the 
carbon  dioxide  which  was  present  in  solution.  But  here  also  the 
supernatant  liquid  is  clear. 

In  herbivorous  animals,  by  which  an  alkaline  urine  is  passed 
habitually,  the  liquid  is  turbid  when  discharged.  In  man  the  pas- 
sage of  a  turbid  urine  is  always  abnormal,  excepting  during  the 
first  days  of  life,  when  cloudy  urine  is  the  rule.  This  is  largely 
.  referable  to  desquamated  epithelial  cells  and  relatively  large  amounts 
of  urates. 

While  the  urine  of  all  mammalian  animals  is  liquid,  the  lower 
animals  excrete  a  urine  that  is  more  or  less  solid.  In  birds 
and  reptiles,  for  example,  in  which  the  ureters  end  in  a  common 
cloaca  with  the  rectum,  the  excrements  appear  in  the  form  of  a  whit- 
ish   pasty  material.     A  gelatinous  urine  is  observed  in  turtles. 

The  color  of  the  urine  in  man  normally  varies  from  light  yellow 
to  dark  amber,  and  is  largely  influenced  by  the  concentration  of  tlie 
secretion  and  its  reaction.  Acid  urine  is  thus  always  darker  than  an 
alkaline  urine,  and  the  color  is  naturally  lighter  when  the  secretion 
is  abundant  than  when  scanty.  A  gradual  darkening  of  the  urine 
is  observed  when  the  material  is  kept  for  some  time  and  access  of 
micro-organisms  is  prevented. 

Deviation  from  the  normal  color  is  notably  observed  in  disease,  or 
following  the  administration  of  various  drugs,  but  may  also  occur  m 


GENERAL  CHARACTERISTICS  OF  THE   URINE.  229 

apparently  healthy  individuals  in  consequence  of  certain  abnormali- 
ties of  metabolism  (see  page  281).  The  urine  may  then  be  of  a 
normal  color  when  recently  passed,  but  soon  darkens  on  standing, 
and  finally  appears  almost  black.  In  diabetes  a  light  color  may  be 
associated  with  a  high  specific  gravity. 

The  odor  of  recently  passed  urine  is  peculiarly  aromatic,  and  is 
probably  referable  to  the  presence  of  several  volatile  acids.  Decom- 
posing urine  has  a  characteristic  odor,  which  is  in  part  due  to 
ammonia. 

Amount. — The  amount  of  urine  eliminated  in  the  twenty-four 
hours  is  quite  variable  even  under  normal  conditions.  It  is,  of 
course,  primarily  dependent  upon  the  amount  of  water  ingested,  but 
is  also  influenced  by  the  character  and  the  quantity  of  the  food,  the 
process  of  digestion,  the  blood-pressure,  the  surrounding  tempera- 
ture, the  emotions,  sleep,  exercise,  body-weight,  sex,  age,  etc.  It 
must  hence  differ  in  different  countries,  according  to  the  habits  of 
the  people,  the  climate,  etc.,  and  we  accordingly  find  that  different 
observers  give  different  figures.  In  Germany  and  Austria,  where 
much  beer  is  consumed,  from  1500  to  2000  c.c.  are  regarded  as 
average  amounts.  In  England  1000  to  1500  c.c.  are  regarded  as 
normal ;  in  France,  1250  to  1300  c.c. 

In  this  country  I  have  found  that  the  average  daily  amount  is 
somewhat  lower,  and  am  inclined  to  regard  an  elimination  of  from 
1000  to  1200  c.c.  as  normal  for  men,  while  in  women  a  somewhat 
smaller  quantity  is  normally  passed.  Children  pass  absolutely  less 
but  relatively  more  urine,  as  compared  with  their  body-weight,  than 
adults. 

In  the  summer-time,  when  the  sweat-glands  are  especially  active, 
and  when  larger  amounts  of  water  are  eliminated  through  the  lungs 
and  the  skin,  the  secretion  of  urine  is  proportionately  less,  but  rarely 
falls  below  800  c.c.  unless  active  exercise  is  indulged  in  at  the  same 
time. 

During  repose,  moreover,  much  less  urine  is  voided  than  when 
exercise  is  taken,  and  we  hence  find  a  smaller  secretion  of  urine 
during  the  night  than  during  the  day.  The  maximum  secretion  is 
usually  observed  a  few  hours  after  the  midday  meal. 

Artificially,  the  secretion  can  be  increased  by  the  ingestion  of 
those  articles  of  food  which  tend  to  increase  the  blood-pressure,  such 
as  coffee,  tea,  and  alcohol.  Many  drugs  also  bring  about  the  same 
effect,  though  the  modus  operandi,  of  each  is  not  known.  The  most 
important  medicinal  diuretics  are  digitalis,  squill,  broom,  juniper, 
nitrous  ether,  urea,  etc.  Distilled  water  also  has  distinct  diuretic 
properties. 

In  disease,  and  notably  in  diabetes  mellitus,  diabetes  insipidus, 
and  chronic  interstitial  nephritis,  the  amount  of  urine  may  far  sur- 
pass the  usual  quantity,  and  may  indeed  exceed  10,000  c.c.  in  the 
twenty-four  hours  (polyuria).  Abnormally  small  amounts,  on  the 
other  hand  (oliguria),  are  observed  in  the  acute  febrile  diseases,  in 


230  THE   VRIXE. 

various  diseases  of  the  circulatory  apparatus,  in  certain  diseases 
of  the  kidneys  and  liver,  etc.     Complete  anuria  may  indeed  occur. 

Specific  Gravity. — The  specific  gravity  of  the  total  amount 
passed  in  twenty-four  hours  normally  varies  between  1.015  and 
1.025.  Generally  speaking,  it  increases  with  the  solids,  the  amount 
of  water  remaining  the  same,  and  diminishes  as  the  amount  of  fluid 
increases  while  the  solids  remain  constant.  Under  pathological 
conditions,  however,  deviations  from  this  rule  are  not  uncommon. 
The  specific  gravity  may  then  fall  as  low  as  1.000  and  1.002,  or  may 
be  increased  to  1.050  and  even  higher. 

Reaction. — The  reaction  of  the  twenty-four  hours'  urine  is,  in 
man,  normally  acid,  sometimes  amphoteric,  and  more  rarely  alka- 
line. The  normal  acidity  is,  however,  not  due  to  the  presence  of  a 
free  acid,  but  to  acid  salts,  and  in  the  first  instance  to  acid  phos- 
phates. As  the  reaction  of  the  blood  is  alkaline,  the  question 
naturally  arises  :  How  is  it  that  an  acid  secretion  can  be  derived 
from  an  alkaline  fluid?  In  the  case  of  the  gastric  juice  we  have 
met  with  a  very  similar  phenomenon,  and  it  was  explained  that  the 
free  acid  in  that  case  most  likely  resulted  through  a  mass-action,  on 
the  part  of  carbonic  acid,  upon  sodium  chloride  within  the  oxyntic 
cells,  the  hydrochloric  acid  being  then  secreted  into  the  lumen  of 
the  glandular  ducts,  while  the  resulting  alkaline  carbonate  is 
returned  to  the  blood.  Similar  conditions  probably  exist  in  the 
kidneys,  where,  as  has  been  mentioned,  the  mineral  salts  are  also 
secreted  into  the  uriniferous  tubules  through  the  specific  activity  of 
the  renal  epithelial  cells.  AVe  may  imagine  that  here  also  a  mass- 
action  on  the  part  of  carbonic  acid  takes  place,  which  in  tiiis  case, 
however,  is  directed  toward  the  alkaline  phosphates  of  the  blood,  as 
is  shown  in  the  equation  : 

Na^HPO^  +  H2CO3  =  NaHjPO^  +  NaHCOs. 

We  may  then  imagine  that  the  resulting  alkaline  carbonate  is 
returned  to  the  blood,  while  the  acid  phosphate  appears  in  the 
urine. 

The  acidity  of  the  urine,  however,  is  primarily  due  to  the 
character  of  the  diet.  In  man  and  the  carnivorous  animals  this 
is  especially  rich  in  albumins,  and  contains  a  comparatively  small 
amount  of  alkaline  salts  or  of  organic  acids  which  could  be  trans- 
formed into  alkaline  carbonates  in  the  body.  During  the  process 
of  metabolism,  then,  the  ingested  albumins  are  broken  down,  and 
uric  acid,  hippuric  acid,  phenaceturic  acid,  oxalic  acid,  aromatic  oxy- 
acids,  and  notably  sulphuric  acid  and  phosphoric  acid  result.  These 
acids,  however,  are  immediately  transformed  into  neutral  salts  by 
combining  with  the  available  alkaline  carbonates  which  are  present 
in  the  lymph  and  the  blood.  As  a  consequence  the  alkalinity  of 
the  l)lood  must  of  necessity  diminish.  But  as  such  a  change  would 
give  rise  to  serious  disturbances,  and  as  there  is  a  strong  tendency 
on  the  part  of  the  body  to  maintain  the  composition  of  the  blood, 


GENERAL   CHARACTERISTICS  OF  THE   URINE.  231 

and  particularly  its  alkalinity,  constant,  a  loss  of  alkali  is  guarded 
against  by  subjecting  the  various  neutral  salts  to  the  specific  activity 
of  the  renal  epithelial  cells.  As  a  result  a  portion  of  the  alkali  is 
returned  to  the  blood,  and  acid  salts  hence  appear  in  the  urine. 

In  the  herbivorous  animals,  on  the  other  hand,  in  which  a  super- 
abundance of  alkaline  salts  is  either  directly  ingested  or  is  formed 
within  the  body  from  salts  of  organic  acids  which  have  been  taken 
with  the  food,  an  alkaline  urine  is  eliminated.  In  this  case  a 
formation  of  acid  salts  and  a  return  of  alkali  to  the  blood  are 
unnecessary.  Similar  conditions  at  times  occur  in  man,  and  the 
elimination  of  an  alkaline  urine,  the  alkalinity  being  due  to  a 
fixed  alkali,  cannot  hence  be  regarded  as  pathological.  During  the 
process  of  digestion,  indeed,  when  an  additional  amount  of  alkaline 
salts  finds  its  way  into  the  blood  in  consequence  of  the  formation 
of  hydrochloric  acid,  an  increased  alkalinity  would  result.  This, 
howev^er,  is  prevented  by  the  excretion  of  a  urine  which,  if  not 
alkaline,  is  at  least  less  acid. 

It  has  been  stated  above  that  the  organic  acids  which  are  formed 
during  the  nitrogenous  metabolism  of  the  body  combine  with  the 
alkaline  carbonates  of  the  lymph  and  blood-plasma  to  form  neutral 
salts.  This  statement  requires  modification  in  so  far  as  it  conveys 
the  idea  that  the  acids  in  question  are  eliminated  in  the  urine  in 
combination  with  fixed  alivalies  only.  As  a  matter  of  fact,  this  is 
true  only  in  part,  and  a  certain  proportion  of  the  acid  is  eliminated 
in  combination  with  ammonia. 

Generally  speaking,  the  ammonium  salts  which  are  formed  within 
the  body  appear  in  the  urine  as  urea,  but  aside  from  their  impor- 
tance in  this  respect  they  represent  a  reserve  of  alkali  which  is  capa- 
ble of  preventing  an  undue  diminution  in  the  alkalinity  of  the  blood 
by  vicariouslv  taking  the  place  of  the  fixed  alkalies.  This  vicarious 
action  is  normally  also  at  work,  but  is  then  comparatively  insig- 
nificant in  extent.  If,  however,  a  specially  large  demand  is  made 
upon  the  alkalies  of  the  body,  as  when  mineral  acids  are  ingested 
for  experimental  purposes,  the  vicarious  action  of  the  ammonium 
salts  at  once  enters  into  play.  Unless  carried  to  extremes,  the 
alkalinity  of  the  blood,  in  the  carnivorous  animals  at  least,  remains 
constant,  but  the  elimination  of  urea  is  proportionately  less,  and  the 
deficit  of  nitrogen  in  this  form  appears  as  ammonia  in  combination 
with  acids. 

By  gradually  increasing  the  amount  of  acid  it  is  thus  possible  to 
bring  about  the  almost  complete  disappearance  of  urea  from  the 
urine.  A  point,  however,  is  finally  reached  when  the  animal  suc- 
cumbs to  acid  intoxication,  and  then,  and  not  before,  may  free  acids 
appear  in  the  urine.  Death  in  such  cases  results  from  suffocation, 
as  there  is  not  sufficient  alkali  left  in  the  lymph  and  plasma  to  com- 
bine with  the  carbon  dioxide  in  the  tissues  (see  page  ^65). 

Conversely,  it  is  possible  to  cause  the  ammonia  to  disappear  from 
the  urine  by  the  administration  of  a  sufficiently  large  quantity  of 


232  THE   UEINE. 

alkali,  and  as  a  consequence  an  increase  in  the  amount  of  urea 
occurs  directly  jjroportionate  to  the  amount  of  ammonia  formerly 
present.  In  herbivorous  animals,  in  which  such  a  vicarious  action 
is  never  necessary  under  normal  conditions,  it  is  accordingly  but 
little  developed,  and  they  hence  soon  die  even  after  the  administra- 
tion of  comparatively  small  amounts  of  mineral  acids. 

It  has  been  stated  that  the  acid  reaction  of  the  urine  is  essentially 
due  to  the  presence  of  acid  phosphates.  Besides  the  acid  phosphates 
normal  urine,  however,  contains  a  certain  amount  of  neutral  phos- 
phates, and  it  may  hap})en  that  both  are  present  in  equal  })roportion. 
But  as  the  neutral  ph(jsj)hates  show  an  alkaline  reaction,  a  neutral 
point  cannot  be  reached,  and  such  urines  hence  color  red  litmus- 
paper  blue,  and  the  blue  paper  red  ;  in  other  words,  they  are  ampho- 
teric. Such  a  reaction  is  not  infrequently  observed,  but  is,  of  course, 
an  accidental  occurrence. 

When  allowed  to  stand  exposed  to  the  air,  every  urine  undergoes 
ammoniacal  decomposition.  This  is  owing  to  the  action  of  certain 
micro-organisms  upon  urea,  which  is  decomposed,  with  the  forma- 
tion of  ammonia,  water,  and  carbon  dioxide,  as  shown  in  the 
equations : 

(1)  CO(NH2)2  +  2H,0  =  (NHJ.COa. 

(2)  (NHJ^COa  =  2NH3  +  H,0  +  CO,. 

The  action  is  thus  a  hydrolytic  decomposition,  and  is  referable  to 
the  activity  of  a  special  ferment,  which  is  found  in  the  micro-organ- 
isms in  question,  notably  the  Mioococcus  urese  and  the  Bacterium 
ureae. 

As  a  result  of  the  presence  of  free  anunonia,  the  soluble  phosphates 
of  the  alkaline  earths  are  then  precipitated  as  tricalcium  phosphate 
and  as  ammonio-magnesium  phosphate,  and  the  soluble  urates  are  at 
the  same  time  transformed  into  the  insoluble  ammonium  salt. 

At  times  an  increase  in  the  acidity  of  the  urine  is  observed  on 
standing,  and  is  generally  ascribed  to  a  peculiar  acid  fermentation 
of  contained  alcohol,  traces  of  carbohydrates,  and  the  like.  More 
often,  however,  a  decrease  in  the  acidity  occurs,  even  though  micro- 
organisms are  absent.  This  is  owing  to  a  decomposition  of  neutral 
urates  by  the  acid  phosphate  of  sodium.  Acid  urates  thus  result, 
and  may  be  further  decomposed,  with  the  liberation  of  uric  acid. 
Both  urates  and  uric  acid  are  then  thrown  down  in  consequence  of 
the  diminished  acidity  of  the  fluid,  and  they  are  hence  no  longer 
capable  of  influencing  the  reaction.  The  changes  which  here  take 
place  may  be  represented  by  the  equations  : 

( 1)  NaH2P0,        +  C^HoXa^N^Oj  =  Na.,HPO,  -f  CjHaNaNA- 

(2)  CjHsNaNA  +  NnH.PO,        =  Na^HFO,  +  CgH.NA- 

As  the  reaction  of  the  urine  is  dependent  in  the  first  instance  upon 
the  character  and  the  quantity  of  the  food  ingested,  viz.,  the  amount 


GENERAL   CHARACTERISTICS  OF  THE   URINE.  233 

of  albumins  and  alkaline  salts  present,  or  of  salts  which  can  be  trans- 
formed within  the  body  into  alkaline  carbonates,  it  follows  that  a 
highly  acid  urine  must  also  result  when  an  increased  destruction  of 
tissue-albumins  is  taking  place  from  whatever  cause.  We  accord- 
ingly find  a  very  acid  urine  in  various  pathological  conditions,  nota- 
bly in  fevers. 

An  alkaline  urine  will  similarly  result  when,  as  in  pneumonia  and 
in  diseases  in  which  large  accumulations  of  fluid  occur  in  the 
serous  cavities  of  the  body,  and  where  a  certain  amount  of  alka- 
line salts  has  thus  been  withdrawn  from  the  circulation,  absorption 
subsequently  occurs.  Alkaline  salts  are,  however,  retained  from 
the  ingested  food,  and  an  increased  elimination  occurs  when  the  addi- 
tional supply  finds  its  way  into  the  plasma  from  these  various  sources. 
A  notable  eiiange  in  the  normal  alkalinity  of  the  blood  can  hence 
scarcely  occur  so  long  as  a  sufficient  amount  of  alkali  is  furnished  in 
the  food. 

In  order  to  decide  whether  the  alkaline  reaction  of  a  specimen  of 
urine  is  due  to  the  presence  of  fixed  or  volatile  alkali,  a  strip  of  red 
litmus-paper  is  clamped  in  the  cork  of  the  bottle,  and  so  arranged  as 
not  to  touch  the  liquid.  If  free  ammonia  is  present,  the  red  color 
changes  to  blue,  while  fixed  alkali  is  indicated  only  when  the  paper 
comes  into  contact  with  the  urine. 

Determination  of  the  Acidity  of  the  Urine. — As  the  acidity  of  the 
urine  is  almost  exclusively  due  to  the  presence  of  acid  phosphates,  its 
determination  resolves  itself  into  the  estimation  of  these  salts.  The 
resulting  values  are  expressed  in  terms  of  hydrochloric  acid,  of  which 
102.8  mgrms.  correspond  to  100  mgrms.  of  the  diacid  sodium  salt. 
Negative  values  are  similarly  expressed  in  terms  of  sodium 
hydrate. 

Freund's  Method. — The  total  amount  of  phosphoric  acid  is  first 
determined  as  described  on  page  238).  In  a  second  portion  the 
monacid  phosphates  are  then  estimated  as  follows :  50  c.c.  of 
urine  are  precipitated  with  a  normal  solution  of  barium  chloride,  10 
c.c.  being  added  for  every  100  mgrms.  of  the  total  amount  of  phos- 
phoric acid  tliat  has  been  found.  The  mixture  is  diluted  with  water 
to  100  c.c,  filtered,  and  the  phosphoric  acid  determined  in  50  c.c.  of 
the  filtrate.  But  as  barium  chloride  precipitates  not  only  the  mon- 
acid phosphate,  but  also  a  small  amount  of  the  normal  phosphates, 
with  the  simultaneous  formation  of  a  small  amount  of  diacid  phos- 
phates, which  latter  pass  into  solution,  an  error  is  thus  incurred. 
This,  however,  remains  constant,  and  amounts  to  3  per  cent,  in  favor 
of  the  diacid  phosphates.  It  is  deducted  from  the  latter,  and  the 
total  amount  of  acid  salts  is  then  determined  by  calculation.  The 
result  is  expressed  in  terms  of  hydrochloric  acid. 

If  relative  values,  on  the  other  hand,  are  desired,  the  percentage 
of  the  diacid  salts  is  ascertained  and  compared  with  the  total  amount 
of  phosphoric  acid,  as  shown  in  the  following  example: 

The  total  amount  of  urine  is  2000  c.c,  and  the  total  amount  of 


234  THE    URINE. 

phosphoric  acid  in  terms  of  P2O5  is  7.72  j^ramraes,  Avhile  the  corre- 
sponding amount  of  acid  phosphates  is  6.736  grammes  after  correct- 
ing as  above  indicated.  The  percentage  of  the  acid  phosphates,  as 
compared  with  the  total  PjO^,  would  then  be  87.2,  as  is  seen  from 
the  calculation  : 

7.72  :  100  :  :  6.7.%  :  x. 

Chemical  Composition  of  the  Urine. — A  general  idea  of  the 
chemical  composition  of  the  urine  and  the  quantitative  variations  of 
the  individual  components  may  be  formed  from  the  accompanying 
table,  which  I  have  constructed  from  numerous  analyses  made  in 
my  laboratory.  The  individuals  from  which  the  urine  was  obtained 
were  all  adults,  and  in  their  general  mode  of  life,  as  regards  diet, 
exercise,  etc.,  followed  the  common  habits  of  the  average  American 
city-dweller. 

Analysis  of  Urine. 

Water 1200-1700  grammes. 

Solids 60.0 

Inorganic  solids 25.0  -26.0  " 

Sulphuric  acid  (H2SO J 2.0-2.5 

Phosphoric  acid  (P2O5) 2.5  -  3.5  " 

Chlorine  (XaCl) 10.0  -15.0 

Potassium  (K,0) 3.3  " 

Calcium  (CaO) 0.2-0.4 

Magnesium  (MgO) 0.5  " 

Ammonia  (NH3) 0.7  " 

Fluorides,  nitrates,  etc 0.2  " 

Organic  solids 20.0  -35.0  " 

Urea 20.0  -30.0  " 

Uric  acid 0.2-1.0 

Xanthin  bases 1.0  " 

Kreatinin 0.05-  0.08 

Oxalic  acid 0.05 

Conjugate  sulphates 0.12-  0.25  " 

Hippuric  acid 0.65-  0.7  " 

Volatile  fatty  acid 0.05  " 

Other  organic  solids 2.5  " 

THE  INORGANIC   CONSTITUENTS  OF  THE    URINE. 

The  inorganic  constituents  of  the  urine  represent  the  excess  of 
mineral  salts  which  find  their  way  into  tlie  ]>l()od  from  the  digestive 
tract,  or  which  develop  within  the  body  during  the  decomposition 
of  the  albumins.  As  has  been  indicated,  they  are  eliminated 
through  the  specific  activity  of  the  renal  epithelial  cells,  so  that 
the  composition  of  the  blood  always  remains  constant.  We  accord- 
ingly find  that  the  ingestion  of  large  amounts  of  food  invarial)]y 
leads  to  an  increased  elimination  of  salts,  and  that  conversely 
smaller  amounts  are  excreted  when  smaller  amounts  are  ingested. 
This  is  true  more  especially  of  the  chlorides  and  the  phosphates, 
while  the  sulphates  are  largely  referable  to  albuminous  destruction, 
and  are  only  ingested  as  such  in  minimal  quantities.  As  the  chlo- 
rides, moreover,  are  far  more  abundant  in  food-stuifs  than  the  phos- 


THE  INORGANIC  CONSTITUENTS  OF  THE   URINE.         235 

phates,  any  variations  from  the  normal  will  affect  these  particularly. 
Under  various  pathological  conditions,  where  a  deficient  amount  of 
food  and  of  inorganic  salts  is  ingested,  or  where  a  considerable 
amount  of  the  salts  is  removed  from  the  circulation,  as  in  conse- 
quence of  hemorrhages,  the  formation  of  exudates  and  transudates, 
etc.,  smaller  amounts  are  accordingly  eliminated,  and  it  may  happen, 
indeed,  that  the  chlorides  disappear  from  the  urine  altogether.  It 
is  noteworthy,  moreover,  that  an  arrest  in  the  elimination  of  the 
chlorides  may  then  also  occur  even  though  a  fair  amount  of  salt  is 
introduced  with  the  food.  In  such  an  event  we  must  assume  that  a 
retention  is  taking  place  in  the  body,  in  consequence  of  tlie  fact  that 
the  lost  fluid,  with  its  various  inorganic  constituents,  is  gradually 
being  replaced.  Subsequently,  when  absorption  of  an  exudate  or  a 
transudate  takes  place,  the  inorganic  solids  find  their  way  into  the  gene- 
ral circulation,  but  are  at  once  eliminated,  as  they  are  present  in  excess. 

The  phosphates  and  sulphates  are  likewise  diminished  under  the 
conditions  just  mentioned,  but  do  not  disappear  entirely,  as  they  are 
in  part  derived  from  the  albumins,  which  are  constantly  undergoing 
destruction  during  the  nitrogenous  metabolism  of  the  body.  The 
diminution  in  the  amount  of  the  phosphates,  however,  exceeds  that 
of  the  sulphates,  as  a  small  fraction  only  of  the  former  is  due  to 
this  source,  while  the  latter  are  largely  derived  from  the  disinte- 
grated albumins. 

The  tenacity  with  which  the  body  maintains  the  normal  composi- 
tion of  the  blood  is  also  well  shown  if  the  chlorides  are  gradually 
diminished  in  the  food,  and  if  their  elimination  from  the  blood  is 
stimulated  by  the  copious  ingestion  of  diuretics.  A  point  is  soon 
reached  when  the  salt  in  question  no  longer  appears  in  the  urine, 
beyond  traces.  If  at  this  stage  the  blood  is  examined,  it  will 
be  found  that  the  amount  of  chlorides  is  practically  the  same  as 
under  normal  conditions.  There  is  a  limit  to  this  power  of  re- 
taining the  mineral  salts,  however,  and  if  the  chlorides  are  withheld 
for  a  length  of  time  and  diuresis  remains  active,  a  gradual  loss 
occurs  nevertheless,  and  in  time  results  in  the  death  of  the  animal. 
It  appears,  however,  that  it  is  not  the  loss  of  chlorine  which  the 
body  tends  to  prevent,  but  that  the  sodium  is  the  component  which 
is  of  prime  importance.  This  becomes  apparent  when  the  potassium 
salt  is  substituted  for  the  sodium  compound,  when  the  same  reten- 
tion of  sodium  chloride  occurs,  while  the  potassium  salt  is  eliminated 
in  the  urine.  In  this  case,  also,  death  ultimately  results  from  what 
is  very  improperly  termed  "  chlorine-hunger," 

If  at  the  stage  when  the  chlorides  have  practically  disappeared 
from  the  urine  salt  is  added  to  the  diet,  a  partial  retention  of  this 
occurs  until  the  original  equilibrium  has  been  restored.  After  that 
a  normal  elimination  is  again  observed,  and  the  amount  then  ex- 
creted practically  corresponds  to  the  quantitv  ingested. 

These  remarks  also  hold  good  for  the  phosphates  and  stdphates  of 
the  body,  though  with   certain  restrictions. 


236  THE    URINE. 

The  bases  which  arc  found  in  the  r.rine  in  combination  with  hydro- 
chloric acid,  sulphuric  acid,  and  j)hos])horic  acid,  are  soduuu,  potas- 
sium, calcium,  magnesium,  and  ammonium.  The  latter,  however, 
occurs  only  in  the  urine  of  man  and  carnivorous  animals.  Calcium 
and  magnesium  occur  almost  exclusively  as  phosphates,  both  of  the 
monacid  and  the  diacid  type.  Traces,  however,  no  doubt  exist  in 
combination  with  hydrochloric  acid  and  sulphuric  acid  as  well,  but 
the  greater  portion  of  these  two  acids,  as  also  of  the  phosphoric  acid, 
is  found  in  the  form  of  sodium  and  potassium  salts.  The  ratio 
between  the  two  latter  is  usually  placed  at  3  : 5,  in  favor  of  sodium. 
It  is  believed  that  the  monacid  phosphates  of  the  alkaline  earths, 
and  notably  the  calcium  salts,  are  held  in  solution  owing  to  the 
presence  of  sodium  chloride  and  the  diacid  phosphates  of  the  alkalies, 
to  which  the  acidity  of  the  urine  is  due. 

The  alkaline  phosphates  normally  exceed  the  earthy  phosphates 
by  one-third,  and  it  is  to  be  noted  that  the  latter  are,  in  part  at 
least,  also  eliminated  by  the  mucous  membrane  of  the  large  intes- 
tine. It  consequently  follows  that  estimation  of  the  ratio  between 
the  two  forms  of  phosphates  can  only  be  of  value  when  the  amount 
which  is  thus  excreted  is  known.  Practically,  such  a  determination 
is,  however,  impossible,  as  a  variable  amount  of  earthy  phosphates 
— in  fact,  the  greater  portion  of  that  which  has  been  ingested — is 
directly  eliminated  through  this  channel. 

While  the  greater  portion  of  the  sulphuric  acid  which  results  from 
the  destruction  of  albumins  within  the  tissues  of  the  body  is  found 
in  the  urine  in  combination  with  inorganic  bases  only,  a  variable 
fraction  also  occurs  united  with  certain  aromatic  substances  which 
are  formed  during  intestinal  putrefaction.  The  resulting  bodies  are 
spoken  of  as  conjugate  or  ethereal  sulphates,  and  normally  repre- 
sent about  one-tenth  of  the  total  amount  of  sulphuric  acid  that 
appears  in  the  urine.  They  comprise  the  alkaline  salts  of  phenol, 
indoxyl,  and  skatoxyl,  and  will  be  considered  later. 

The  mineral  and  conjugate  sulphates  together  are  s])oken  of  as 
the  "acid"  sulphur  of  the  urine,  in  contradistinction  to  the  so-called 
neutral  sulphur,  which  represents  a  variable  fraction  that  escapes 
oxidation  in  the  body  and  finds  its  way  into  the  urine  as  such.  This 
comprises  such  substances  as  thiosulphuric  acid,  tauro-carbaminic 
acid,  sulphocyanic  acid,  cystin,  cystein,  ethyl  sulphide,  uroferric 
acid,  alloxyproteinie  acid,  etc.  They  are  described  in  detail  at 
another  place. 

In  addition  to  the  salts  mentioned,  a  variable  amount  of  carbo- 
nates is  found  in  every  urine.  In  man  and  the  carnivorous  ani- 
mals this  is  usually  small ;  but  in  the  herbivorous  animals  large 
quantities  are  normally  found,  and  the  alkaline  reaction  of  such 
urine  is  indeed  largely  referable  to  this  source.  The  acid  occurs  in 
combination  with  the  alkalies  and  the  alkaline  earths,  and  owing  to 
the  presence  of  the  latter  especially  the  urine  of  such  animals  is 
normally  turbid. 


THE  INORGANIC  CONSTITUENTS  OF  THE   URINE.         237 

Of  other  inorganic  constituents,  every  urine  also  contains  iron 
(partly  in  organic  combination),  silicates,  fluorides,  hydrogen  per- 
oxide, and  nitrates,  all  of  which,  however,  are  present  only  in 
traces.  The  nitrates  are  probably  introduced  with  vegetable  food, 
and  disappear  from  the  urine  during  starvation.  During  ammo- 
niacal  fermentation  they  are  reduced  to  nitrites,  and  later  disappear. 

The  quantitative  variations  of  the  inorganic  constituents  of  human 
urine  are  shown  in  the  following  table  : 

Chloriiles  I  calculated  as  HCl) 6.2-9.4  grammes. 

Phosphates  (calciihiled  as  Pp^) 2.5-.''..0 

Sulphates  (calculated  as  11,804) 2.0-2.5         " 

Sodium    (calculated    as    Na.p) 4.0-G.O         " 

Potassium  (calculated  as  K.p)  2.0-3.0         " 

Ammonium  (calculated  as  NH^) 0.7     gramme. 

Magnesium  (calculated  as  MgO) 0.5--0.6         " 

Calcium  (calculated  as  CaO)      0.2-0.4        " 

Quantitative  Estimation  of  the  Mineral  Ash. 

Ten  c.c.  of  urine  are  placed  in  a  weighed  crucible  and  evaporated 
at  a  temperature  of  about  100°  C.  The  crucible  is  then  covered 
with  its  lid  and  carefully  heated  over  a  small  flame  until  the  organic 
matter  has  been  carbonized  and  fumes  are  no  longer  evolved.  On 
cooling,  the  residue  is  extracted  with  boiling  water.  The  washings 
are  passed  through  a  small  filter,  the  weight  of  the  ash  of  which  is 
known,  and  the  filter,  together  with  the  carbonaceous  residue,  is 
incinerated  until  a  white  ash  is  obtained.  This  process  may  be 
aided,  if  necessary,  by  moistening  the  material  with  a  little  alcohol 
or  water.  The  washings  are  then  placed  in  the  crucil)le  and  evapo- 
rated at  100°  C.  The  residue  is  finally  dried  in  a  hot-air  bath, 
heated  until  the  bottom  of  the  crucible  just  turns  red,  and  is  then 
allowed  to  cool  over  sulphuric  acid  and  weighed.  The  weight  of  the 
mineral  ash  of  the  10  c.c.  of  urine  is  then  ascertained  by  deducting 
that  of  the  crucible  and  the  ash  of  the  filter. 

Quantitative  Estimation  of  the  Chlorides. 

The  chlorides  of  the  urine  are  most  conveniently  estimated  accord- 
ing to  the  method  of  Salkowski-Volhard.  To  this  end,  10  c.c.  of 
urine  are  diluted  with  50  c.c.  of  distilled  water,  and  treated  with  4 
c.c.  of  concentrated  nitric  acid  and  15  c.c.  of  a  standard  solution  of 
silver  nitrate.  The  mixture  is  further  diluted  to  100  c.c,  thor- 
oughly agitated,  and  passed  through  a  dry  filter.  In  a  carefully 
measured  portion  of  the  filtrate  the  excess  of  silver  is  then  titrated 
with  a  solution  of  potassium  sulphocyanide  of  such  strength  that  25 
c.c.  correspond  to  10  c.c.  of  the  silver  solution.  A  few  drops  of  a 
saturated  solution  of  ammonio-ferric  alum  serve  as  indicator.  The 
amount  of  silver  solution  used  in  the  precipitation  of  the  chlorides 
in  the  10  c.c.  of  urine  is  then  calculated.  The  number  of  cubic 
centimeters  which   was   necessary  for  this  purpose,  multiplied  by 


238  THE    URINE. 

0.01,  indicates  the  amount  of  chlorides  present   in   the    10  c.c.  of 
urine,  calcukited  as  sodium  salt. 

The  presence  of  albumins  and  sugar  does  not  interfere  \\'\i\\  the 
method. 

Quantitative  Estimation  of  the  Phosphates. 

To  determine  the  amount  of  the  alkaline  and  earthy  phosphates 
together,  50  c.c.  of  urine  are  treated  with  5  c.c.  of  a  solution  con- 
taining about  100  grammes  of  sodium  acetate  and  100  c.c.  of  a  30 
per  cent,  solution  of  acetic  acid  to  the  liter.  In  this  manner  any 
monacid  phosphates  that  may  be  present  are  transformed  into  diacid 
phosphates.  A  few  drops  of  tincture  of  cochineal  are  then  added, 
and  the  mixture  heated  to  the  boiling-jjoint  and  titrated  with  a 
standard  solution  of  uranyl  acetate  or  nitrate  until  a  greenish  color 
is  noticed  in  the  resulting  precipitate  of  uranyl  phos])hate  which 
does  not  disappear  on  stirring.  From  the  number  of  cubic  centim- 
eters employed  the  corresponding  amount  of  phosphates  is  then 
determined  in  terms  of  P2O5.  The  uranium  solution  is  of  such 
strength  that  20  c.c.  represent  0.1  gramme  of  P2O5. 

The  presence  of  sugar  and  albumins  does  not  interfere  M'ith  the 
method. 

Separate  Estimation  of  the  Earthy  and  Alkaline  Phosphates. 

Two  hundred  c.c.  of  urine  are  rendered  strongly  alkaline  with 
ammonia  and  set  aside  for  several  hours.  The  earthy  phosphates 
are  thus  precipitated,  and  are  collected  on  a  small  filter,  washed  with 
dilute  ammonia  (1  :  3),  transferred  to  a  beaker,  and  dissolved  with 
as  little  acetic  acid  as  possible.  Distilled  water  is  added  so  as  to 
make  the  volume  of  the  liquid  about  50  c.c,  when  the  solution  is 
boiled  and  titrated  as  above.  In  a  second  portion  of  the  urine 
the  total  amount  of  phosphates  is  then  determined.  The  difference 
between  the  two  results  indicates  the  amount  of  phosphates  which 
is  present  in  combination  with  alkalies. 

If  it  is  desired  to  remove  the  total  phosphates  from  a  specimen 
of  urine  preliminary  to  some  further  step  in  analysis,  the  fluid  is 
rendered  alkaline  with  the  hydrate  of  an  alkaline  earth  and  pre- 
cipitated with  a  soluble  calcium  or  barium  salt.  Or  we  may  pre- 
cipitate directly  with  neutral  or  basic  acetate  of  lead.  In  the  first 
instance,  the  excess  of  calcium  or  barium,  and  in  the  second,  that 
of  lead,  must  then  be  removed. 

Quantitative  Estimation  of  the  Sulphates. 

To  determine  the  amount  of  both  mineral  and  conjugate  sulphates, 
100  c.c.  of  urine  are  treated  with  8  c.c.  of  strong  hydrochloric  acid 
and  heated  to  the  boiling-point.     In  this  manner  the  conjugate  sul- 


THE   ORGANIC  CONSTITUENTS  OF  THE   URINE.  239 

phates  are  decomposed,  with  the  liberation  of  the  sulphuric  acid, 
which  is  then  precipitated,  together  with  the  mineral  sulphates,  as 
barium  sulphate.  To  this  end,  20  c.c,  of  a  hot  saturated  solution  of 
barium  chloride  are  added  to  the  hot  liquid.  The  mixture  is  ke})t 
on  a  boiling  water-bath  until  the  precipitate  has  settled,  when  it  is 
collected  on  a  small  filter,  washed  with  boiling  water,  then  with  hot 
alcohol,  and  finally  with  ether.  After  incineration  the  lilter  ash  is 
deducted  from  the  total  weight.  The  result  may  be  expressed  in 
terms  of  H.,SO^,  of  SO3  or  S,  by  multiplying  the  weight  of  the  barium 
sulphate  by  0.42015,  0.34301,  or  0.13744,  respectively. 

To  determine  the  amount  of  mineral  sulphates  and  of  conjugate 
sulphates  separately,  100  c.c.  of  urine  are  treated  with  an  equal 
volume  of  an  alkaline  solution  of  barium  chloride,  which  consists  of 
two  volumes  of  a  solution  of  barium  hydrate  and  one  volume  of  the 
chloride,  both  saturated  at  ordinary  temperatures.  The  mineral  sul- 
phates are  thus  precipitated  together  with  the  phosphates  and  are 
filtered  oiF.  One  hundred  c.c.  of  the  filtrate,  corresponding  to  50 
c.c.  of  the  urine,  are  now  strongly  acidified  with  hydrochloric  acid 
and  boiled.  The  conjugate  sulphates  are  decomposed,  and  the  lib- 
erated sulphuric  acid  is  thrown  down,  as  above.  The  process  is 
then  continued  as  described.  The  resulting  value  represents  the 
amount  of  sulphuric  acid  which  was  present  in  combination  with 
phenol,  indoxyl,  and  skatoxyl.  By  deducting  this  value  from  the 
total  amount  of  sulphuric  acid  the  mineral  portion  is  ascertained. 

Test  for  Nitrates. — To  demonstrate  the  presence  of  nitrates, 
200  c.c.  of  urine  are  treated  with  30  to  40  c.c.  of  chemically  pure, 
concentrated  sulphuric  acid  or  hydrochloric  acid,  and  distilled  upon 
a  sand-bath.  The  distillate  is  received  into  a  dilute  solution  of 
caustic  alkali.  Owing  to  the  presence  of  reducing  substances  in  the 
urine,  the  nitric  acid  is  thus  transformed  into  nitrous  acid,  and 
passes  over  as  such.  The  presence  of  nitrites  may  then  be  demon- 
strated as  usual. 

THE  ORGANIC  CONSTITUENTS  OF  THE  URINE. 

The  organic  constituents  of  the  urine  comprise  the  normal  end- 
products  of  the  nitrogenous  metabolism  of  the  body,  various  products 
of  albuminous  putrefaction  which  have  found  their  way  into  the 
general  circulation  from  tiie  intestinal  canal,  and  certain  pigments 
which  are  more  or  less  intimately  related  to  the  normal  blood-pig- 
ment. In  addition,  traces  of  various  other  substances  may  be 
encountered,  the  origin  of  which  is  obscure.  Under  pathological 
conditions  we  meet  with  certain  normal  constituents  of  the  blood 
which  generally  do  not  appear  in  the  urine  as  such,  or  occur  in 
infinitesimally  small  amounts,  and  also  with  various  abnormal 
products  of  metabolism,  all  of  which  will  be  considered  in  detail. 


240  THE   URISE. 

The  Nitrogenous  Constituents  of  the  Urine. 
Urea. 

While  in  birds  mid  reptiles  the  greater  portion  of  the  urinary 
nitrogen  is  excreted  in  the  form  of  uric  acid,  urea  constitutes  the 
most  important  end-product  of  the  nitrogenous  metabolism  of  the 
remaining  groups  of  vertebrate  animals.  In  man,  8G  per  cent,  of  the 
total  nitrogen  eliminated  in  the  urine  appears  in  this  form. 

Origin. — Formerly  it  was  supposed  that  urea  resulted  from  uric 
acid  through  a  process  of  oxidation,  and  that  this  was  its  only 
source.  We  have  seen  that  the  formation  of  urea  from  uric  acid  is 
possible,  and  we  cannot  deny  that  a  certain  proportion  of  the  sub- 
stance may  be  derived  in  this  manner.  ]Modern  researches,  how- 
ever, have  shown  that  in  man  and  the  mammalian  animals  uric  acid 
is  largely  derived  from  a  destruction  of  the  nucleins  within  the 
body,  and  results  from  oxidation  of  the  xanthin  bases,  which  are 
thus  set  free.  In  birds  and  reptiles,  on  the  other  hand,  the  greater 
portion  of  the  uric  acid  is  formed  synthetically  from  simpler  sub- 
stances, and  is  hence  not  directly  comparable  to  the  form  which 
is  found  in  the  higher  animals.  In  these  a  synthetic  formation  is 
also  possible,  but  probably  does  not  occur  under  normal  conditions. 
As  we  can  therefore  recognize  one  origin  of  uric  acid  only  in  the 
mammal,  and  as  this  source  of  the  nitrogen  is  insignificant  when 
compared  with  the  large  amount  of  urea  actually  found,  we  are 
forced  to  the  conclusion  that  the  greater  j)ortion  of  the  urea  must 
originate  in  a  different  way. 

It  has  l)een  repeatedly  shown  that  during  the  decomposition  of 
the  albumins  by  means  of  acids  and  alkalies,  as  also  during  the 
process  of  tryptic  digestion  and  albuminous  putrefaction,  a  large 
amount  of  mono-amido-acids  results.  It  has  hence  been  supposed 
that  these  bodies  probably  represent  intermediary  products  in  the 
transformation  of  the  albuminous  nitrogen  into  uroa,  and  it  has 
actually  been  demonstrated  that  in  mammals — and  to  these  I  shall 
confine  my  remarks  for  the  present — the  administration  of  such 
acids  in  the  food  is  followed  by  a  corresponding  increase  in  the 
amount  of  urea.  Under  certain  pathological  conditions,  moreover, 
these  acids  appear  in  the  urine  as  such,  and  it  is  then  noted  that  the 
elimination  of  urea  is  much  diminished.  In  health,  however,  this 
does  not  occur,  and  on  examination  of  the  difix'rent  tissues  and 
organs  of  the  body  such  acids  are  found  only  in  traces.  We  must 
hence  assume  that  these  acids,  supposing  them  to  occur  as  primary 
products  of  albuminous  decomposition  within  the  body,  are  trans- 
formed at  once  into  other  substances,  which  in  turn  give  rise  to  urea. 
As  all  these  bodies  on  oxidation  yield  ammonium  carbonate,  this 
substance  would  hence  suggest  itself  as  a  probable  antecedent  of 
urea.  We  find,  as  a  matter  of  fact,  that  ammonium  carl)onate 
when  ingested  by  the  mouth,  or  otherwise  introduced  into  the  body, 


THE  ORGANIC  CONSTITUENTS   OF  THE    URINE.  241 

appears  in  the  urine  as  urea.     This  transformation  of  mono-amido- 
acids  into  urea  may  be  represented  by  the  following  equations : 

(1)  CH2(NH2).COOH  +  20  =  H.COONH,  +  CO^ 

Glycocoll.  Ammonium 

formate. 

(2)  2H.C00NH^  +  20  =  (NHJ^COs  +  Hp  +  00, 

(3)  (NH,),.C03  =  CO<  +  2H,0 

Urea. 

Drechsel  has  further  shown  that  the  amido-acids  yield  carbamic 
acid  on  oxidation,  and  that  through  alternate  oxidation  and  reduction 
urea  can  result  from  the  ammonium  salt,  as  shown  in  the  equation : 

/NH^  .NH2 

0C<  =  OCC  +  H2O 

That  carbamic  acid  is  present  in  the  normal  acid  urine  of  man 
and  the  dog  has  been  proved.  Nencki  and  Hahn,  moreover, 
observed  that  in  dogs  in  which  the  liver  was  temporarily  excluded 
from  the  general  circulation  larger  amounts  of  carbamic  acid 
appeared  in  the  urine  than  under  normal  conditions,  and  that  the 
animals  showed  symptoms  of  intoxication  identical  with  those 
observed  when  carbamates  are  directly  introduced  into  the  blood- 
current.  These  symptoms  were  also  present  when  carbamates  were 
introduced  into  the  stomach,  in  which  case  normal  dogs  show  no 
signs  of  poisoning. 

While  it  has  been  assumed  above,  that  urea  is  largely  referable  to 
a  transformation  of  mono-amido-acids  into  ammonium  carbonate  or 
carbamate,  as  the  case  may  be,  and  while  it  has  been  shown  that 
such  a  transformation  actually  does  occur,  we  must  yet  remember 
that  only  traces  of  amido-acids  are  normally  found  in  the  tissues. 
There  is  reason  to  believe  that  the  greater  portion  of  the  albuminous 
nitrogen  is  normally  set  free  from  the  various  organs  of  the  body  in 
the  form  of  the  ammonium  salt  of  paralactic  acid,  and  there  is  a 
tendency  among  physiologists  at  the  present  time  to  regard  this 
salt  as  the  common  antecedent  of  urea.  It  has  been  demonstrated, 
as  a  matter  of  fact,  that  urea  results  when  ammonium  lactate  is 
passed  through  the  isolated  liver  of  a  dog ;  and  clinically,  also,  we 
observe  that  both  ammonia  and  lactic  acid  appear  in  the  urine  in 
increased  amounts  when  the  liver  is  extensively  diseased.  Similar 
results  are  obtained  in  birds,  in  which  uric  acid  represents  the 
principal  end-product  of  nitrogenous  metabolism.  In  geese  it  is 
thus  noted  that  after  extirpation  of  the  liver  the  greater  portion  of 
the  urinary  nitrogen  appears  in  the  form  of  ammonium  lactate. 

Under  normal  conditions  it  is  assumed  that  the  lactate  is  trans- 
formed into  ammonium  carbonate,  which  in  turn  yields  the  carba- 
mate, and  the  urea  finally  results  through  a  synthetic  process,  which 
is  probably  effected  through  the  agency  of  a  certain  ferment.  We 
may  further  imagine  that  the  paralactic  acid  in  the  last  instance  may 

16 


242  THE  URINE. 

result  from  a  decomposition  of  the  raono-amido-radicles  of  the  albu- 
minous molecules,  and  in  this  form  the  theory  would  embrace  the 
two  outlined  abo\'e.  The  various  changes  may  be  represented  by 
the  equations  : 

(1)  2NH4.C3HA  +  120  =  (NHJ^COj  +  5C0j  +  5H,0 
Ammonium  lactate. 

(2)  (NH4)2C03  =  C0<  +   H,0 

\O.NH, 

Ammouium 
carbamate. 

,NH,  .NH„ 

(3)  C0(  =  C0(  +   H,0 

\O.NH4  ^NH, 

Urea. 

While  we  have  seen  that  urea  may  originate  in  the  animal  body 
through  a  process  of  oxidation  only,  as  also  synthetically  through 
alternate  reductions  and  oxidations,  tliere  is  still  another  possibility, 
viz.,  that  it  may  be  derived  from  the  albumins  by  hydrolysis  only. 
We  know,  as  a  matter  of  fact,  that  a  number  of  nitrogenous  substances 
are  found  in  the  body,  such  as  kreatin,  krcatinin,  oxaluric  acid,  and 
others,  which  on  hydrolytic  decomposition  give  rise  to  the  formation 
of  urea,  and  it  is  quite  possible  tliat  a  certain  proportion  may  be 
referable  to  this  source. 

We  have  also  seen  that  on  hydrolytic  decomposition  all  albumins 
Avhich  have  been  examined  in  tliis  direction  yield  com])aratively 
large  amounts  of  arginin,  and  that  this  can  be  further  decomposed 
in  the  same  manner  into  ornithin  and  guanidin,  which  latter  then 
yields  urea.  As  arginin  is  now  known  to  occur  in  the  animal  body 
as  such,  there  is  no  reason  for  supposing  that  a  certain  fraction  of 
the  urea  may  not  be  formed  as  just  indicated,  and  Drechsel  indeed 
has  expressed  the  opinion  that  10  per  cent,  of  the  total  amount  may 
thus  result  through  hydrolytic  processes  only. 

Hoppe-Seyler  has  suggested  that  in  the  transformation  of  the 
mono-amido  acids  into  urea  cyanic  acid  may  be  produced  as  an  inter- 
mediary product,  and  that  urea  then  results  through  the  interaction 
of  two  molecules  of  this  acid,  as  is  shown  in  the  equation  : 

CONH   +   CONH   +   HjO  =   C0<  +   COj. 

In  all  ]irobability  a  certain  amount  of  urea  is  produced  in  the 
animal  body  in  different  ways,  and  there  is  reason  to  believe,  more- 
over, that  its  formation  is  not  confined  to  one  single  organ.  The 
greater  portion,  no  doubt,  is  formed  synthetically  in  the  liver.  Of 
this,  indeed,  we  have  abundant  proof.  It  has  been  shown  that  in 
diseases  of  this  organ  which  are  associated  with  an  extensive  destruc- 
tion of  the  glandular  elements  a  diminished  amount  of  urea  is  found 
in  the  urine,  while  ammonia  and  lactic  acid  are  present  in  increased 
quantity,  and  the  mono-amido  acids  may  appear  as  such.     In  cases 


THE   ORGANIC  CONSTITUENTS   OF  THE   URINE.  243 

of  this  kind  as  nnicli  as  37  ])er  cent,  of  the  total  amount  of  urinary 
nitrogen  has  been  found  in  the  form  of  ammonia.  In  the  mammal, 
moreover,  symptoms  of  carbamic  acid  poisoning  are  observed  when 
the  liver  is  excluded  from  the  general  circulation,  and,  as  has  been 
shown,  the  formation  of  urea  from  ammonium  lactate  or  carbonate 
may  be  demonstrated  in  the  isolated  livers  of  dogs.  As  negative 
results  were  obtained  by  von  Schroeder  when  blood  containing  am- 
monium carbonate  was  passed  through  the  kidneys  and  through  the 
isolated  hind-quarters  of  dogs,  the  conclusion  suggests  itself  that  in 
these  organs  a  formation  of  urea  does  not  occur.  This  inference  is, 
however,  not  admissible  in  the  light  of  our  modern  knowledge  of 
the  origin  of  urea,  for  we  can  readily  conceive  that  although  a  syn- 
thetic formation  from  ammonium  carbonate  may  not  occur  in  these 
organs,  it  is  nevertheless  possible  that  it  may  originate  in  a  different 
manner. 

The  transfusion  experiment,  after  all,  only  shows  whether  or  not  a 
new  body  can  be  formed  in  the  organ  under  investigation  from  other 
substances  which  are  passed  through  it  as  such.  Before  deciding 
that  urea  cannot  be  produced  in  these  parts  it  would  hence  be  neces- 
sary to  ex]»eriment  with  all  those  substances  Avhich  can  be  made  to 
yield  urea  in  the  test-tube,  and  which  we  know  to  occur  in  the  ani- 
mal body.  In  the  spleen,  where  arginin,  for  example,  is  known  to 
occur,  it  would  be  of  interest  to  ascertain  whether  urea  is  produced 
here  when  blood  containing  arginin  is  passed  through  the  organ. 

If  we  accept  the  modern  doctrine  that  urea  not  only  originates  in 
the  animal  body  in  different  ways,  but  that  it  may  also  be  formed  in 
other  organs  besides  the  liver,  we  can  also  understand  why  it  is  that 
in  certain  diseases  of  the  liver  the  diminution  in  the  formation  of 
urea  is  not  always  proportionate  to  the  extent  of  parenchymatous 
degeneration,  and  that  no  case  has  been  reported  in  which  the 
formation  of  urea  ceased  altogether.  It  is  also  made  clear  why 
urea  is  found  in  the  urine  of  birds  and  reptiles,  although  a  synthetic 
production  of  the  substance  manifestly  does  not  occur  in  these 
animals.  Its  origin  is  here  no  doubt  to  be  sought  in  its  formation 
from  such  bodies  as  kreatin,  kreatinin,  arginin,  and  the  like. 

Nitrogenous  Equilibrium. — The  albumins,  of  course,  are  the  ulti- 
mate source  of  the  urea.  According  to  Pettenkofer,  they  exist  in 
the  body  in  two  forms,  viz.,  as  organized  albumin  which  is  built  up 
into  tissues,  and  as  so-called  circulating  albumin  which  is  present  in 
excess  of  what  is  actually  required,  and  is  bi'oken  down  directly 
and  eliminated  in  the  urine  without  having  entered  into  the  con- 
struction of  the  body  proper.  This  portion  of  the  albumin  furnishes 
the  greater  portion  of  the  urea,  while  the  organized  albumin  repre- 
sents a  minor  but  more  constant  source.  The  actual  amount  that  is 
eliminated  is  thus  primarily  dependent  upon  the  amount  ingested. 

The  total  urinary  nitrogen  is  under  normal  conditions  practically 
equivalent  to  the  quantity  ingested,  barring  the  small  fraction  which 
escapes  digestion  in  the  feces.     Such  a  condition  is  spoken  of  as  the 


244  THE   URINE. 

nitrogenous  equilibrium  of  the  body.  Of  this,  liowever,  different 
levels  may  exist,  which  may  vary  in  the  same  individual.  If  the 
amount  of  nitrogenous  food  is  thus  diminished,  tlie  amount  of 
nrinary  nitrogen  will  also  decrease  ;  and  if  the  amount  of  food  then 
remains  constant,  the  nitrogenous  output  will  likewise  remain  the 
same.  If,  on  the  other  hand,  more  nitrogen  is  now  ingested,  an 
increased  elimination  will  result ;  but  a  certain  fraction  is  retained 
by  the  body,  and  gradually  a  higher  level  of  equilibrium  becomes 
established. 

There  are  natural  limits  to  this  power  of  accommodation,  how- 
ever, and  we  finally  reach  a  point  which  varies  in  different  indi- 
viduals, where  a  further  increase  in  the  amount  of  nitrogen  that  is 
ingested  does  not  lead  to  a  higher  level  of  equilibrium,  and  where 
consequently  a  further  retention  of  nitrogen  does  not  occur.  Over- 
feeding then  results  in  various  digestive  disturbances — diarrhoea  and 
vomiting  occur,  and  the  body  thus  protects  itself  against  an  undue 
accumulation  of  circulating  albumin  which  it  would  not  be  able  to 
dispose  of  in  a  normal  manner. 

Underfeeding,  on  the  other  hand,  gradually  leads  to  an  increased 
destruction  of  the  organized  albumins.  For  a  while  the  reserve  of 
fats  and  carbohydrates  is  still  capable  of  protecting  the  body  against 
an  unduly  rapid  loss  of  nitrogen  from  this  source,  but  death  finally 
results. 

From  the  fact  that  the  level  of  nitrogenous  equilibrium  is  different 
in  different  people  and  may  vary  in  one  and  the  same  individual,  it 
follows  that  the  amount  of  urea  also  must  vary.  Any  figures  indi- 
cating the  amount  of  urea  that  is  eliminated  in  the  urine  can  there- 
fore he  of  little  value  unless  we  are  acquainted  with  the  actual  state 
of  health  of  the  individual,  his  body-weight,  his  habits  of  life  as 
regards  exercise,  the  amount  of  nitrogenous  food  ingested,  etc. 
Having  a  knowledge  of  all  these  factors,  however,  we  may  be  able 
to  say  whether  the  amount  of  urea  is  normal  or  not.  Certain  figures 
have  ])een  given  by  physiologists  to  indicate  the  amount  of  nitrogen- 
ous food  which  should  enter  into  the  composition  of  the  diet,  and 
from  these  we  can  approximately  calculate  the  amount  of  urea  that 
should  appear  in  the  urine.  By  estimating  this  in  turn,  or  still 
better,  of  course,  the  total  amount  of  nitrogen,  we  can  accordingly 
decide  whether  or  not  the  individual  is  consuming  a  sufficient  amount 
of  nitrogen  in  his  food.  The  figures,  however,  have  been  constructed 
without  due  regard  to  the  factors  above  indicated,  and  are  in  my 
opinion,  at  least,  too  high  as  averages. 

I  am  willing  to  admit  that  an  elimination  of  40-50  grammes  of 
urea  may  be  normal  in  certain  cases,  as  in  soldiers  on  forced  marches, 
among  the  lal)oring-classes,  etc.,  but  I  should  certainly  look  upon 
the  average  merchant  or  student,  who  leads  a  sedentary  life,  as  an 
overfed  individual  if  his  daily  elimination  of  urea  should  exceed  30 
grammes  in  the  twenty-four  hours.  Among  the  well-to-do  classes 
I  find  that  an   elimination  of  from  20  to  25  grammes  is  probably 


THE  ORGANIC  CONSTITUENTS  OF  THE   URINE.  245 

normal,  taking  tlie  body-weight  of  the  person  into  consideration. 
A  smaller  amount  even  is  not  infrequently  met  with  in  individuals 
of  sedentary  habits  who  are  in  perfect  health,  but  I  should  scarcely 
regard  such  a  quantity  as  normal  for  the  average  laboring-man. 
While  extensive  variations  in  the  amount  of  urea  are  thus  observed 
in  health,  still  greater  deviations  from  average  figures  are  noted  in 
disease,  but  here  as  there  we  must  always  take  into  account  the 
amount  of  nitrogen  that  is  ingested  and  the  body  weight. 

An  increase  in  the  elimination  of  urea,  referable  to  the  destruction 
of  organized  albumins,  is  here  frequently  observed,  but  may  be 
obscured,  owing  to  a  deficient  ingestion  of  nitrogen,  unless  the  amount 
of  the  latter  is  known.  At  this  place,  however,  it  is  scarcely  neces- 
sary to  enter  into  pathological  considerations,  and  I  must  refer  the 
reader  to  other  works  for  detailed  information  on  such  questions. 
But  I  may  briefly  recall  that  in  certain  diseases  of  the  liver  in 
which  an  extensive  destruction  of  the  parenchyma  is  taking  place 
the  amount  of  urea  may  be  greatly  diminished,  although  a  fairly 
abundant  supply  of  nitrogenous  food  is  ingested.  As  has  been 
shown,  the  synthetic  formation  of  urea  is  here  seriously  impeded, 
and  as  a  result  we  find  that  a  considerable  proportion  of  the  urinary 
nitrogen  then  appears  in  the  form  of  ammonium  salts  of  paralactic 
acid,  of  carbaraic  acid  and  carbonic  acid,  and  in  extreme  cases, 
indeed,  mono-amido  acids,  such  as  leucin  and  tyrosin,  may  be  found. 

Properties  of  Urea. — Urea  crystallizes  in  two  forms,  viz.,  in 
long,  fine  white  needles  if  rapidly  formed,  or  in  long,  colorless 
rhombic  prisms  when  allowed  to  crystallize  more  slowly  from  its 
solutions. 

It  melts  at  130°  to  132°  C,  but  is  probably  decomposed  already 
at  a  temperature  of  100°  C.  It  is  readily  soluble  in  water  and 
alcohol,  but  is  insoluble  in  anhydrous  ether,  chloroform,  and  benzol. 
As  the  substance  is  an  acid  amide,  its  solutions  present  a  neutral 
reaction. 

In  accordance  with  its  character  as  an  unsaturated  amide  of 
carbonic  acid,  however,  it  combines  with  acids  to  form  crystalline, 
salt-like  compounds.  The  most  important  of  these  are  the  nitrate 
and  the  oxalate. 

Urea  nitrate,  CO(NH2)2.HN03,  crystallizes  in  two  forms,  viz.,  in 
delicate  rhombic,  horizontal  platelets,  which  are  commonly  arranged 
overlapping  in  a  shingle-like  manner  when  rapidly  formed,  or  as 
thicker  rhombic  columns  or  plates  when  allowed  to  crystallize  more 
slowly. 

Urea  nitrate  is  readily  soluble  in  distilled  water,  but  dissolves 
with  difficulty  if  this  is  acidulated  with  nitric  acid,  and  also  in 
alcohol.  Its  formation  is  frequently  observed  when  urine  contain- 
ing much  urea  is  examined  for  albumin  in  the  cold  with  nitric 
acid.  On  standing,  the  nitrate  may  then  separate  out  in  crystalline 
form.  On  heating,  the  substance  is  decomposed  without  leaving  a 
residue. 


246  THE   URLXE. 

Urea  oxalate,  CO(NH2)2.C2H^Oi,  crystallizes  in  rh()nil)ic  plates,  or 
hexagonal  prisms,  and  is  less  soluble  in  water  than  the  nitrate;  in 
alcohol  and  in  dilute  solutions  of  oxalic  acid  it  is  nearly  inst)luble. 
The  substance  is  obtained  in  crystalline  form  on  adding  a  saturated 
solution  of  oxalic  acid  to  a  concentrated  solution  of  urea. 

Urea  also  combines  with  various  neutral  salts,  such  as  sodium 
chloride  and  ammonium  chloride,  and  also  with  the  nitrates  of 
sodium  and  the  oxides  of  silver  and  mercury,  to  form  double  salts. 
With  mercuric  nitrate  three  diiferent  compounds  result,  according 
to  the  concentration  of  the  two  solutions,  viz.,  CO(NH2)2.Hg2(N03)4, 
CO(NH2)2.Hg3(N03)„  and  [CO(NH2),]2.Hg(N03)2  +  3HgC).  The 
latter  compound  is  of  special  interest,  as  Liebig's  quantitative  esti- 
mation of  urea,  which  was  formerly  much  employed,  was  based 
upon  its  formation.  It  results  when  a  2  per  cent,  solution  of  urea 
is  treated  with  a  feebly  acid  solution  of  mercuric  nitrate,  and  the 
mixture  is  subsequently  neutralized. 

]\Iercuric  chloride  precipitates  urea  in  alkaline,  but  not  in  neutral 
solutions. 

Very  important,  further,  is  the  behavior  of  urea  toward  sodium 
hypochlorite  or  hypobromite,  as  the  most  usual  method  of  esti- 
mating urea  in  the  clinical  laboratory  is  based  upon  the  reaction 
which  here  takes  place.  This  reaction  may  be  represented  by  the 
equation  : 

CO(NH2)2  +  SNaOBr  =  3NaBr  +  COj  +  2H2O  +  2N. 

In  practical  work  an  alkaline  solution  of  the  hypobromite  is 
employed,  so  that  the  carbon  dioxide  which  is  liberated  is  at  once 
absorbed,  while  the  nitrogen  remains.  It  is  to  be  noted,  however, 
that  while  1  gramme  of  urea  should  theoretically  give  rise  to  the 
formation  of  372.7  c.c.  of  nitrogen,  354.3  c.c.  are  at  best  obtained 
at  0°  C  and  a  pressure  of  760  Hgmm,  In  clinical  work  this 
difference  is  unimportant,  and  it  is  in  a  measure  equalized  by  the 
evolution  of  a  small  amount  of  nitrogen  from  some  of  the  other 
nitrogenous  constituents  which  are  at  the  same  time  present.  On 
hydrolysis  urea  is  transformed  into   ammonium   carbonate : 

CO(NH2)2  +  2H2O  =  {^N'H^),CO,. 

This  occurs  during  the  process  of  ammoniacal  fermentation,  which 
results  when  urine  is  exposed  to  the  air,  and  is  referable,  as  I  have 
pointed  out,  to  the  action  of  a  specific  bacterial  enzyme.  On  boiling 
with  acids  or  alkalies  the  same  result  is  primarily  obtained,  but  the 
salt  is  then  further  decomposed,  with  the  liberation  of  carbon  dioxide 
and  ammonia,  respectively.  This  flecomposition  is,  however,  also 
noted  during  the  process  of  ammoniacal   fermentation. 

On  heating  the  substance  in  aqueous  solution  in  a  sealed  tube  to 
a  temperature  of  100°  C.  ammonia  and  carbon  dioxide  likewise 
result. 


THE  ORGANIC  CONSTITUENTS  OF  THE   URINE.  247 

Nitrous  acid  when  added  in  excess  decomposes  urea,  with  the 
formation  of  nitrogen,  carbon  dioxide,  and  water,  but  the  acid  is  at 
the  same  time  decomposed,  as  is  seen  in  the  equation  : 

CO(NH2)2  +  2HNO2  =  CO2  +  4N  +  3HA 

This  reaction  is  utilized  when  it  is  desired  to  remove  nitrous  acid 
from  a  solution. 

On  heating  the  dry  substance  in  a  test-tube  to  a  temperature  of 
about  150°-170°  C,  fumes  of  ammonia  are  freely  evolved,  owing  to 
the  decomposition  of  the  urea  with  the  formation  of  biuret,  as  shown 
in  the  equation  : 

/NH, 
C0< 
2CO(NH2)2  =  NH3  +    ^    >NH 

Biuret. 

On  further  heating,  more  ammonia  is  given  off;  the  melted  mass 
finally  solidifies,  and  may  be  shown  to  contain  both  biuret  and 
cyanuric  acid.  The  reaction  which  takes  place  may  be  represented 
as  follows  : 

(1)  3CO(NH2)2   =   3C0NH  +  3NH3 
Cyanic  acid. 

(2)  3C0NH  =  CgNgfOri);, 
Cyanuric  acid. 

To  demonstrate  the  presence  of  the  biuret,  the  residue  is  dis- 
solved in  a  dilute  solution  of  sodium  hydrate,  when  upon  the  care- 
ful addition  of  a  dilute  solution  of  copper  sulphate  a  beautiful, 
purple-red  color  develops. 

A  very  delicate  test  also  is  the  following :  2  c.c.  of  a  concentrated 
solution  of  furfurol  are  treated  with  4-6  drops  of  strong  hydro- 
chloric acid.  If  to  this  mixture,  which  should  not  present  a  red 
color,  a  small  crystal  of  urea  is  then  added,  a  deep  violet  develops 
in  the  course  of  a  few  minutes. 

Synthetic  Formation. — As  has  been  mentioned,  urea  was  the  first 
organic  substance  formed  in  the  animal  body  which  was  made  syn- 
thetically in  the  chemical  laboratory.  Wohler  in  1828  produced  the 
substance  artificially  by  evaporating  an  aqueous  solution  of  ammo- 
nium cyanate,  when  a  rearrangement  of  atoms  occurs  and  urea  results  : 

NH, 

(NH4)CN0  =  COC 

Other  methods  now  exist  by  which  urea  can  also  be  made  syn- 
thetically, but  they  are  all  more  or  less  modifications  of  the  one  just 
described. 

Isolation  from  the  Urine. — To  isolate  urea  on  a  small  scale,  50-100 
c.c.  of  urine  are  evaporated  to  a  syrup  on  a  water-bath  and  ex- 
tracted with    150   c.c.   of  strong  alcohol   by  rubbing    in    a    mortar. 


248  THE   URINE. 

The  alcoholic  extract  is  filtered,  the  alcohol  distilled  off,  and  the 
syrupy  residue  treated  with  concentrated  nitric  acid  in  the  cold. 
The  urea  nitrate  which  crystallizes  out  on  standing  is  filtered 
off  with  a  suction-pump,  dissolved  in  hot  water,  and  the  aqueous 
solution  decolorized  by  gently  heating  with  animal  charcoal.  The 
colorless  filtrate  is  then  treated  with  barium  carbonate  in  substance 
so  long  as  carbon  dioxide  is  evolved,  and  finally  rendered  alkaline 
with  barium  hydrate  solution.  The  urea  is  thus  liberated  according 
to  the  equation  : 

2CO(NHa),.HIs'03  +  BaCOj  =  BalNOa)^  +  2CO(NH2)2  +  H^O  +   CO^. 

The  solution  is  now  evaporated  to  dryness  and  the  residue  extracted 
with  absolute  alcohol.  On  concentrating  this  extract  the  urea  crj's- 
tallizes  out  in  colorless  prisms,  which  may  then  be  treated  as  above 
indicated. 

Quantitative  Estimation. — In  the  clinical  laboratory  the  old  method 
of  Knop  and  Hiifnor  is  almost  exclusively  employed.  This  is  based 
upon  the  decomposition  of  urea  Avith  sodium  hypobromite  in  alkaline 
solution,  as  already  described.  The  nitrogen  which  is  thus  liber- 
ated is  measured  and  the  corresponding  amount  of  urea  determined 
by  calculation. 

For  scientific  purposes,  however,  this  method  in  any  one  of  its 
numerous  modifications  is  not  sufficiently  accurate,  as  the  actual 
volume  of  nitrogen  which  is  obtained  always  falls  somewhat  short 
of  the  theoretical  amount.  The  sodium  hypobromite,  moreover, 
also  causes  a  partial  decomposition  of  other  nitrogenous  constituents 
of  the  urine,  and  as  the  resulting  amount  of  nitrogen  is  not  con- 
stant, a  further  error  is  incurred.  In  scientific  research  we  are 
hence  forced  to  resort  to  some  other  procedure,  such  as  that 
proposed  by  Morner  and  Sjoquist,  or  the  simpler  method  which  has 
recently  been  suggested  by  Folin. 

Method  of  Morner  and  Sjoquist. — This  is  based  upon  the 
fact  that  the  organic  nitrogenous  constituents  of  the  urine,  with  the 
exception  of  urea  and  ammonia,  are  precipitated  by  an  alkaline 
barium  chloride  mixture.  This  precipitate  is  insoluble  in  ether- 
alcohol,  while  urea  and  ammoniacal  salts  are  dissolved  together  with 
a  small  amount  of  barium  hydrate.  In  the  concentrated  ether- 
alcoholic  filtrate  the  nitrogen  is  then  determined  according  to  Kjel- 
dahl's  method,  after  the  ammoniacal  salts  have  been  decomposed, 
and  the  resulting  ammonia  has  been  driven  off.  From  the  per- 
centage of  nitrogen  the  corresponding  amount  of  urea  is  then  cal- 
culated by  multiplying  by  2.14. 

Five  c.c.  of  urine  are  treated  with  an  equal  volume  of  baryta 
mixture,  which  contains  250  grammes  of  barium  chloride  in  1000 
c.c.  of  a  o  per  cent,  solution  of  barium  hydrate.  To  this  are  added 
100  c.c.  of  a  mixture  of  two  parts  of  97  per  cent,  alcohol  and  one 
part  of  ether.  After  twenty-four  hours  the  precipitate  is  filtered 
off  and  treated  with  100  c.c.  of  ether-alcohol.     Filtrate  and   wash- 


THE   ORGANIC  COySTITUENTS  OF  THE   URINE.  249 

ings  are  then  concentrated  at  a  temperature  not  exceeding  60°  C. 
to  about  20  c.c,  or  to  a  point  where  ammonia  is  no  longer  evolved, 
which  can  be  recognized  by  testing  the  vapor  with  litmus-paper. 
A  small  amount  of  water  may  be  added  if  the  solution  should 
become  too  concentrateil.  It  is  then  washed  into  a  Kjeldahl  digest- 
ing flask  with  as  little  water  as  possible,  to  which  a  few  drops  of 
concentrated  sulphuric  acid  have  been  added.  The  solution  is  now 
concentrated  to  a  very  small  volume,  upon  a  water-bath,  and  treated 
with  20  c.c.  of  a  mixture  of  two  parts  of  concentrated  sulphuric 
acid  and  one  part  of  the  fuming  acid.  A  pinch  of  yellow  oxide  of 
mercury  (about  0.3  gramme)  is  further  added,  when  the  process  is 
continued  according  to  Kjeldahl,  as  described  below. 

Method  of  Folix. — This  is  based  upon  the  following  considera- 
tions :  crystallized  magnesium  chloride,  MgCU-GHjO  boils  in  its 
water  of  crystallization  at  a  temperature  of  about  160°  C.  Urea 
is  quantitatively  decomposed  in  such  a  solution  into  ammonia  and 
carbon  dioxide  within  one-half  hour.  If  the  process  is  carried  out 
in  acid  solution,  the  ammonia  can  subsequently  be  distilled  off  after 
rendering  the  mixture  alkaline,  and  is  then  titrated.  The  cor- 
responding amount  of  urea  is  ascertained  by  calculation.  At  the 
same  time,  however,  the  preformed  ammonia  is  obtained,  and  it  is 
hence  necessary  to  eliminate  this  source  of  error  by  a  separate 
estimation  of  this  form.  This  is  conveniently  done  according  to 
the  method  which  has  likewise  been  suggested  by  Folin  (see  below). 

Method. — Three  c.c.  of  urine  are  placed  in  an  Erlenmeyer  flask 
of  200  c.c.  capacity,  together  with  20  grammes  of  magnesium 
chloride  and  2  c.c.  of  concentrated  hydrochloric  acid.  It  is  neces- 
sary to  measure  the  urine  very  accurately  with  a  pipette  graduated 
in  one-twentieths  c.c.  (The  magnesium  chloride  usually  contains 
a  small  amount  of  ammonia,  which  must  be  separately  determined.) 
The  flask  is  closed  with  a  perforated  stopper  through  which  a 
specially  constructed  safety  tube  passes.^  The  mixture  is  now 
boiled  until  the  dro])s  flowing  back  through  the  tube  produce  a 
hissino-  sound  on  cominon  in  contact  with  the  solution.  After  this 
point  has  been  reached,  the  boiling  is  continued  more  moderately 
for  about  forty-five  minutes.  In  order  to  obviate  immoderate  foam- 
ing a  piece  of  paraffin  about  twice  as  large  as  a  coffee  bean  is 
added.  The  solution  while  still  hot  is  carefully  diluted  to  about 
500  c.c. — at  first  by  allowing  the  water  to  flow  drop  by  drop 
through  the  tube :  it  is  then  transferred  to  a  1000  c.c.  retort, 
treated  with  about  7  or  8  c.c.  of  a  20  per  cent,  solution  of  sodium 
hydrate,  and  the  ammonia  distilled  off  into  a  measured  amount 
of  a  decinormal  solution  of  sulphuric  acid.  The  distillation  may 
be  interrupted  when  about  350  c  c.  have  passed  over  (viz.,  after 
about  sixty  minutes).  The  distillate  is  boiled  for  a  moment  to 
remove  any  carbon  dioxide  which  may  be  present  in  solution,  and 
on  cooling  is  titrated  to  determine  the  excess  of  acid.     Each  cubic 

»  The  tube  can  be  procured  from  Eimer  &  Amend's,  of  New  York. 


250  THE  uniXE. 

centimeter  of  the  decinormal  ammonia  present  in  the  distillate  cor- 
responds to  0.003  gramme,  viz.,  to  0.1  per  cent  of  urea. 

From  this  result  the  amount  of  preformed  ammonia  and  that 
present  in  the  20  grammes  of  magnesium  chloride  must  be  deducted. 

If  desired,  the  estimation  can  also  be  made  with  the  urea-con- 
taining filtrate  obtained  with  Morner  and  Sjoquist's  method,  but 
Folin  states  that  the  previous  isolation  of  the  urea  in  such  manner 
is  probably  not  necessary. 

Estimation  of  the  Preformed  Ammonia  (according  to  Folin). — 
Folin's  most  recent  method  is  described  by  Shaffer  as  follows : 
To  25  or  50  c.c.  of  urine,  placed  in  an  areometer  cylinder  about  45 
cm.  liigh  and  5  cm.  in  diameter,  there  are  added  8  or  10  grammes  of 
sodium  chloride,  according  to  the  amount  of  urine,  5  to  10  c.c.  of 
petroleum  or  toluol,  and  1  or  2  grammes  of  .-odium  carbonate.  The 
sodium  chloride  is  added  to  prevent  decomposition.  A  current  of 
air  is  driven  tluough  the  urine,  carrying  off  the  ammonia  set  free 
by  the  sodium  carbonate ;  this  is  absorbed  by  passing  through  a 
definite  amount  of  j^'^  normal  acid,  the  excess  of  which  is  after- 
ward titrated  and  the  ammonia  calculated.  The  length  of  time 
necessary  for  the  completion  of  the  operation  will  depend  upon  the 
amount  of  air  passing  through  the  liquid,  as  well  as  upon  the 
temperature.  At  22°-25°  C.  and  with  an  air  current  of  approxi- 
mately 700  lilers  per  hour,  the  ammonia  will  be  completely  driven 
off  from  25  c.c.  of  urine  in  an  hour  and  a  quarter,  or  from  50  c.c. 
in  an  hour  and  a  half.  With  a  larger  volume  of  urine,  a  smaller 
air  current,  or  lower  temperatures  of  the  urine,  a  longer  time  will 
be  necessary.  The  requisite  time  must  be  learned  for  each  air 
current.  This  can  easily  be  done  by  continuing  the  operation, 
until  in  an  hour  there  is  no  ammonia  given  off.  It  is  necessary  to 
pass  the  air  through  a  tuft  of  absorbent  cotton,  or  closely  packed 
glass  wool,  after  it  leaves  the  urine  and  before  it  passes  through  the 
acid,  in  order  to  prevent  alkali  from  being  carried  over.  The  air 
current  should  be  uniform  and  constant.  Folin  uses  a  specially 
constructed  tube,  for  the  absorj)tion  of  the  ammonia  by  the  acid. 
Where  this  is  not  done,  it  will  usually  l)e  found  necessary  to  pass 
the  air  through  two  successive  portions  of  acid  to  prevent  loss  of 
ammonia. 

Folin  employs  alizarin  red  as  indicator;  2  drops  of  a  1  per  cent, 
solution  suffice.  The  titration  is  carried  to  the  red  point,  not  to 
violet.  The  method  as  described  is  also  ;q)i)licable  in  the  case  of 
blood. 

Instead  of  this  method,  one  can  also  use  the  Boussingault  vacuum 
distillation  method,  as  modified  l\v  Folin.^ 

Estimation  of  the  Total  Urinary  Nitrogen. — Kjeldahl's  Method. 
— The  method  is  based  upon  the  observation  that  on  treating  urine 
with  a  mixture  of  two  parts  of  concontrated  sulphuric  a-cid  and  one 
part  of  fuming  sulpiiuric  acid  and   boiling,  the  entire  amount   of 

«  For  a  description  of  this  method  see  P.  Shaffer,  "  On  the  Quantitative  Determination  of 
Ammonia  in  the  Urine."    Amer.  Jour,  of  Phys.,  vol.  viii.,  No.  4,  p.  330. 


THE  ORGANIC  CONSTITUENTS  OF  THE   URINE.  251 

nitrogen  can  be  transformed  into  ammonium  sulphate.  This  is 
then  decomposed  with  an  excess  of  sodium  hydrate  and  the  liberated 
ammonia  estimated  In*  distilling  into  a  known  amount  of  dilute  acid, 
and  retitrating  the  excess  of  acid. 

Five  c.c.  of  urine  are  treated  in  a  Kjeldahl  digesting  flask  with 
a  pinch  of  yellow  oxide  of  mercury  (abont  0.3  gramme)  and  20  c.c. 
of  the  sulphuric  acid  solution.  The  mixture  is  boiled  until  a  per- 
fectly colorless  solution  is  obtained.  Vigorous  ebullition,  how- 
ever, must  be  avoided,  and  the  flask  should  be  placed  at  an  angle 
of  about  45  degrees,  so  as  to  prevent  loss  from  spurting.  The  milder 
the  ebullition  the  better.  On  cooling,  the  contents  of  the  flask  are 
transferred  to  a  Kjeldahl  retort,  with  the  aid  of  a  little  distilled  water. 
Sodium  hydrate  solution  (27  per  cent.)  is  then  added  until  the  greater 
portion  of  the  acid  is  neutralized.  The  fluid  is  allowed  to  cool  again, 
and  a  few  pieces  of  granulated  zinc  or  a  little  talcum  is  thrown  in, 
when  a  mixture  of  the  hydrate  solution  and  of  a  4  per  cent,  solution 
of  potassium  sulphide  is  further  added  in  excess.  Of  either  solution, 
40  c.c.  are  added  in  all.  The  addition  of  the  latter  is  necessary,  as 
the  mixture  not  only  contains  ammonium  sulphate,  but  also  amido- 
compounds  of  mercury,  which  latter  would  not  give  up  their  entire 
amount  of  nitrogen  if  sodium  hydrate  alone  were  })resent.  The  talcum 
or  zinc  merely  prevents  an   unduly  violent  bumping  when  boiling. 

Neubertr  has  recently  suo-o-ested  that  it  is  more  convenient  to  add 
about  1  gramme  of  sodium  thiosulphate  to  the  caustic  alkali  solution 
for  every  0.4  gramme  of  oxide  of  mercury,  instead  of  the  sulphide, 
as  the  latter  must  always  be  prepared  anew,  and  as  the  addition  of 
the  corresponding  amount  of  liquid  prolongs  the  distillation.  In 
the  alkaline  solution  the  amido-mercuric  sulphate  is  then  decomposed 
according  to  the  equation  : 

/NH3X 
Hg<^  >S0,  +  Xa,S,03  +  H,0  =  HgS  +  (NH,),SO,  +  Na.SO,. 

The  retort  is  then  immediately  connected  with  a  condenser  through 
the  intervention  of  a  Kjeklahl  distilling  tube.  The  condensing  tube 
dips  into  a  nitrogen  bulb,  which  contains  a  carefully  measured 
amount  of  a  one-fourth  normal  solution  of  sulphuric  acid ;  30  c.c. 
are  usually  sufficient.  Tiie  mixture  is  now  distilled  until  about 
two-thirds  have  passed  over.  The  condenser  is  rinsed  w^ith  a 
little  distilled  water,  which  is  added  to  the  distillate.  After  the 
addition  of  a  few  drops  of  tincture  of  cochineal  the  excess  of 
acid  is  retitrated  with  a  one-fourth  normal  solution  of  sodium 
hydrate.  The  difference  indicates  the  amount  of  acid  which  was 
consumed  in  uniting  with  the  liberated  ammonia.  As  1  c.c.  of  the 
one-fourth  normal  solution  represents  0.0035  gramme  of  nitrogen, 
the  amount  contained  in  the  5  c.c.  of  urine  is  ascertained  by  multi- 
plying the  number  of  cubic  centimeters  employed  by  this  figure, 
from  which  the  total  amount  of  twenty-four  hours  is  then  readily 
calculated.     The  corresponding  amount  of  albumin   is  obtained  by 


252  THE   URINE. 

multiplying  this  figure  by  6.25.  The  method,  as  just  described, 
appears  simple  enougli,  but  in  reality  requires  a  considerable  amount 
of  experience  to  obtain  figures  that  are  reliable.  With  experience, 
however,  the  method  is  exceedingly  accurate.  In  every  case,  of 
course,  chemically  pure  reagents  are  necessary,  and  it  is  well  to  test 
these  with  care  before  proceeding. 

Uric  Acid. 

Whereas  in  mammals,  the  amphibia,  and  fishes  urea  is  the  most 
important  end-product  of  nitrogenous  metabolism,  the  greater  por- 
tion of  the  urinary  nitrogen  in  birds  and  reptiles  is  eliminated  as 
uric  acid. 

Origin. — The  formation  of  uric  acid  in  birds  and  reptiles  is  analo- 
gous to  the  formation  of  urea  in  the  mammal.  It  is  derived  in 
the  last  instance  from  the  albumins  of  the  tissues  and  from  the 
ingested  f)od,  and,  like  urea,  is  formed  synthetically  in  the  liver. 
This  is  true  at  least  of  the  greater  portion  ;  while  a  variable  frac- 
tion originates  from  the  nucleins,  viz.,  the  xanthin  bases.  Organic 
ammonium  salts,  amido-acids,  urea,  and  ammonium  carbonate,  when 
given  to  birds  in  their  food,  appear  in  the  urine  as  uric  acid,  and 
it  is  now  thought  that  here  also  the  greater  portion  of  the  nitrogen 
is  carried  to  the  liver  as  ammonium  lactate.  We  accordingly  find 
that  after  extirpation  of  the  liver  almost  all  the  urinary  nitrogen 
appears  in  this  form,  and  that  ammonium  carbonate  when  given  by 
the  mouth  is  eliminated  as  such.  Of  the  manner  in  which  the 
synthesis  of  uric  acid  is  eifected  in  the  liver,  however,  we  know 
but  little.  Urea  or  ammonium  carbonate  cannot,  of  course,  give 
rise  to  its  formation  alone,  as  the  available  amount  of  carbon  is 
too  small.  W^e  must  hence  assume  that  some  other  substance  enters 
into  the  reaction.  This  substance,  however,  is  as  yet  unknown,  but 
we  may  imagine  that  one  portion  of  ammonium  lactate  is  first  trans- 
formed into  ammonium  carbonate,  and  that  the  uric  acid  is  tiien 
formed  through  the  union  of  this  with  another  molecule  of  lactic 
acid.  Horbaczewski,  indeed,  has  shown  that  artificially  uric  acid 
may  be  formed  from  lactic  acid,  ammonia,  and  carbon  dioxide,  by 
heating  trichloro-lactic  amide  together  with  urea.  The  reaction 
which  takes  place  may  be  represented  by  the  equation  : 

2CO(NHj)2  +  C3CI3O2H2.NH2  =  NH,C1  +  2HC1  +  Hp  +  QH^NA- 
Trichloro-lactic  Uric  acid, 

amide. 

On  the  other  hand,  it  is  possible  that  uric  acid  may  result  through 
the  union  of  glycocoll  and  urea,  and  artificially  this  synthesis  can 
indeed  be  eifected.  We  have  seen,  moreover,  that  on  hydrolytic 
decomposition  uric  acid  yields  annnonia,  carbon  dioxide,  and  gly- 
cocoll. In  this  case  the  resulting  reaction  could  be  expressed  by 
the  equation : 

3CO(NH2),  +  CII,(NH.,).COOII  =  SNH^  +  2H2O  +  CjH.NA- 


THE  ORGANIC  CONSTITUENTS   OF  THE   URINE.  253 

Wiener  has  recently  suggested  that  uric  acid  may  be  formed 
synthetically  through  the  union  of  tartronic  acid  with  two  urea 
radicles,  and  the  intermediate  formation  of  dialuric  acid,  the  tar- 
tronic acid  being  itself  derived  from  lactic  acid  by  oxidation.  This 
transformation  is  represented  by  the  following  equations  : 

NH,      COOII       NH CO 

II  II 

1.  CO   +  CHOH  =  CO         CHOH  +  2H2O 

II  I  II 

NH2       COOH  NH CO 

Urea.      Tartronic  Dialuric  acid, 

acid. 

NH CO  NH CO 

I  I  H,N\  I  I 

2.  CO         CHOH  +     "      >C0  =  CO         C— HN\ 

I  li  HjN^  I  II  /CO  +  2H2O 

NH CO  NH C— HN 

Dialuric  acid.  Urea.  Uric  acid. 

Wiener's  experimental  basis  of  the  synthetic  formation  of  uric 
acid  in  birds  is  quite  convincing  and  accords  well  with  other 
observed  facts. 

While  the  greater  portion  of  the  uric  acid  is  thus  formed  syn- 
thetically in  the  liver  of  birds  and  reptiles,  a  variable  but  much 
smaller  amount  results  directly  from  the  xanthin  bases  through  a 
process  of  oxidation. 

These  are  in  part  derived  from  disintegrating  cells  of  the  body, 
and  are  to  a  certain  extent  also  referable  to  the  ingested  food.  In 
man  and  most  mammals  this  is  indeed  the  only  source  of  the  uric 
acid,  and  through  the  researches  of  Horbaczewski  we  now  know 
that  the  nuclear  uric  acid,  as  we  may  term  it,  is  formed  together 
with  the  xanthin  bases  in  all  organs  of  the  body,  and  is  most 
abundantly  produced  in  those  which  are  especially  rich  in  nuclei, 
such  as  the  spleen  and  the  lymph-glands.  The  very  interesting 
observation  was  further  made  that  larger  amounts  of  uric  acid  could 
be  obtained  from  these  parts  when  the  blood  used  in  the  transfu- 
sion experiments  contained  much  oxygen,  while  with  venous  blood 
xanthin  bases  only  were  produced.  In  the  amphibia  and  fish,  in 
which  the  oxidation-processes  are  especially  sluggish,  we  accordingly 
find  xanthin  bases,  but  little  or  no  uric  acid.  The  interesting  ques- 
tion now  suggests  itself.  Why  is  it  that  in  mammals  uric  acid  appears 
in  the  urine  at  all  in  view  of  the  fact  that  uric  acid  which  is  intro- 
duced into  the  stomach  is  eliminated  as  urea  ?  A  final  answer  to 
this  question  cannot  be  given,  but  there  is  reason  to  suppose  that  the 
uric  acid  is  here  first  carried  to  the  liver,  and  is  probably  oxidized 
to  urea  by  the  oxidizing  ferments  of  this  organ.  We  find,  as  a 
matter  of  fact,  that  an  increased  elimination  of  uric  acid  results  at 
once  when  the  blood  of  the  portal  vein  is  prevented  from  flowing 
through  the  liver  by  establishing  a  so-called  Eck  fistula  between 
this  and  the  inferior  cava,  and  when  the  hepatic  artery  is  at  the 
same  time  ligated.     In  this  manner  the  blood  of  the  spleen  and  the 


254  THE   URINE. 

extensive  lymphatic  districts  of  the  intestinal  tract  is  carried  directly 
into  the  general  circulation,  and  the  coml)ined  xanthin  bases  and 
uric  acid  hence  find  their  way  into  the  urine  without  being  sub- 
jected to  the  action  of  the  oxydases  of  the  liver.  We  may  hence 
conclude  that  the  appearance  of  these  bodies  in  the  urine  is  under 
normal  conditions,  owing  to  the  fact  that  not  all  the  blood  of  the 
bodv  reaches  the  liver  before  being  carried  to  the  kidneys. 

In  birds  and  reptiles  we  have  also  seen  that  a  certain  amount 
of  urea  appears  in  the  urine,  and,  as  I  have  already  explained,  this 
is  no  doubt  produced  directly  in  the  tissues.  As  ingested  urea  is 
here  transformed  into  uric  acid,  we  must  hence  assume  that  the 
portion  which  is  eliminated  in  the  urine  has  reached  the  kidneys 
without  having  previously  passed  through  the  liver,  and  the  process 
is  thus  quite  analogous  to  what  we  observe  in  mammals  in  the  case 
of  uric  acid. 

Under  normal  conditions  the  elimination  of  uric  acid  nirely  ex- 
ceeds 1  gramme,  but  larger  amounts  may  be  encountered  in  disease. 

In  leukaemia  especially,  a  greatly  increased  elimination  is  thus 
commonly  observed,  and  is  here  no  doubt  referable  to  the  increased 
destruction  of  leucocytes.  But  excessive  amounts  of  uric  acid 
mav  also  occur  in  conditions  in  which  there  is  no  direct  evidence 
of  increased  nuclear  destruction.  In  many  cases  of  this  kind  the 
increased  elimination  is  apparently  dependent  upon  the  amount  of 
animal  food  that  is  ingested,  and  it  would  appear  that  in  such  cases 
the  liver  has  lost  to  a  greater  or  less  degree  its  power  of  oxidizing 
the  uric  acid,  which  reaches  it  from  this  source.  Were  this  true,  we 
should  then  also  expect  that  relatively  larger  amounts  of  xanthin 
bases  should  find  their  way  into  the  urine,  and  this  indeed  may 
actually  occur.  But,  on  the  other  hand,  an  increased  elimination  of 
uric  acid  and  xanthin  bases  may  also  be  observed  although  the 
patient  has  been  placed  on  a  diet  Avhich  is  practically  free  from 
nuclear  nucleins.  An  adequate  explanation  of  such  an  occurence  is 
as  yet  wanting.  We  may  here  also  suppose  that  the  liver  has 
lost  its  power  of  oxidation  so  far  as  the  alloxuric  bodies  are  con- 
cerned. But  we  must  bear  in  mind  that  uric  acid  is  formed  in  all 
the  tissues  of  the  body,  and  that  the  relative  amount  which  thus 
originates,  as  compared  with  the  xanthin  bases,  is  largely  influenced 
by  the  intensity  of  the  processes  of  oxidation.  It  is  hence  also 
conceivable  that  in  such  cases  these  may  be  deficient,  while  the 
liver  may  function  in  a  normal  manner.  The  possibility  of  a 
synthetic  production  of  uric  acid,  finally,  may  also  enter  into 
consideration. 

The  question  of  the  nature  of  the  so-called  uric  acid  diathesis  is 
thus  still  ill  statu  quo,  and  the  same  may  be  said  of  the  formation 
of  uratic  deposits  in  the  joints  and  tendons  in  gout.  It  appears, 
however,  that  an  increased  production  of  uric  acid,  contrary  to 
what  was  formerly  supposed,  plays  no  role  in  the  causation  of  the 
latter  disease. 


THE  OEGANIC  COSSTITUENTS   OF  THE   VRINE.  255 

Properties  of  Uric  Acid. — Pure  uric  acid  crystallizes  iu  traus- 
pareut,  colorless  rhombic  platelets,  the  angles  of  which  are  often 
rounded  oif.  Such  crystals  are  at  times  seen  in  urinary  sediments, 
but  more  commonly  the  substance  is  here  found  in  the  form  of 
brownish-yellow  whetstone-like  crystals,  which  may  occur  singly, 
but  are  frequently  arranged  in  groups.  These  are  quite  character- 
istic, and  cannot  be  confounded  with  crystals  of  any  other  substance 
that  may  occur  in  the  urine. 

A  great  many  other  forms  may,  however,  also  be  encountered, 
such  as  dumb-bells,  somewhat  irregular  hexagonal  platelets,  jiaddle- 
shaped  crystals,  etc.,  the  nature  of  which  is  not  at  once  apparent. 

Uric  acid  is  almost  insoluble  in  cold  water  (1  :  40,000),  with 
difficulty  also  in  boiling  water  (1  :  1800),  and  insoluble  in  alcohol 
and  ether. 

Ill  concentrated  sulphuric  acid  and  boiling  glycerin  it  dissolves 
with  comparative  ease  and  without  undergoing  decomposition.  It  is 
a  dibasic  acid,  and  accordingly  combines  with  bases  to  form  neutral 
and  acid  salts.  Of  these,  the  neutral  salts  of  potassium  and 
lithium  are  the  most  soluble,  while  the  acid  salts,  and  notably  acid 
ammonium  urate,  are  quite  insoluble.  Its  compounds  with  the 
alkaline  earths  are  likewise  soluble  only  with  great  difficulty.  In 
the  urine  uric  acid  is  said  to  be  present  as  a  quadriurate,  viz.,  as 
a  hyperacid  compound,  in  which  one  molecule  of  sodium  (viz., 
potassium  or  ammonium)  is  in  combination  with  two  molecules  of 
uric  acid.  Its  solubility  in  the  urine  is  largely  dependent  upon  the 
amount  of  water,  the  reaction,  and  the  presence  of  mineral  salts  and 
possibly  of  pigments.  On  standing,  however,  in  the  absence  of 
micro-organisms,  the  quadriurate  is  decomposed,  with  the  liberation 
of  free  uric  acid  and  acid  biurates,  which  latter  are  then  again  trans- 
formed into  quadriurates  through  the  agency  of  the  diacid  phos- 
phates, and  through  a  repetition  of  this  process  all  the  uric  acid 
finally  separates  out,  as  is  shown  in  the  equations : 

(1)  HNa.a,H,NAC\H,N,03  +  H,0  =  C^H^NA  +  HNa.CjH.NA 

Sodium  quadriurate.  Uric  acid.  Acid  sodium 

biurate. 

(2)  2HNa.C5H2N403  +  NaH^PO,        =-  HNa-QH^NA-CsH^NA  +  Na^HPO^ 

Acid  biurate.  Quadriurate. 

In  the  urine  of  birds  and  reptiles  the  uric  acid  is  said  to  occur 
exclusively  in  the  form  of  quadriurates.  Neutral  urates  are  not 
found  in  the  urine.  Of  the  compounds  which  uric  acid  forms  with 
the  salts  of  the  heavy  metals,  the  silver  and  copper  salts  deserve 
especial  mention,  as  some  of  the  methods  which  are  employed  in  the 
quantitative  estimation  of  the  substance  are  dependent  upon  their 
formation.  The  salts  of  uric  acid  are  readily  decomposed  by  hydro- 
chloric acid,  and  on  standing  the  free  substance  crystallizes  out  from 
the  solution.  The  intimate  relation  which  exists  between  uric  acid 
and  the  xanthin  bases,  as  also  its  character  as  a  diureid,  has  already 
been  considered  (pages  104  and  105). 


256  THE   URINE. 

Tests  for  Uric  Acid. — Murexid  Test. — If  a  few  crystals  of  uric 
acid  arc  evaporated  with  a  few  drops  of  concentrated  nitric  acid  on 
a  porcelain  plate,  a  yellow  or  brick-red  residue  remains.  {3n  cool- 
ino;,  a  droj)  or  two  of  ammonia  are  added,  when  a  iieautiful  purple- 
red  color  develops,  owing  to  the  formation  of  ammonium  purpurate 
(murexid).  If  now  an  excess  of  sodium  hydrate  solution  is  added, 
the  ammonium  salt  is  transformed  into  the  corresponding  sodium 
salt,  and  tlie  purple  red  passes  into  a  bluish  violet.  This  disappears 
on  heating  and  docs  not  return  on  cooling  (compare  with  the  similar 
reaction  of  xanthin  and  guanin). 

Copper  Test. — A  few  crystals  of  uric  acid  are  dissolved  in 
sodium  hydrate  solution  and  treated  with  a  few  drops  of  Fehling's 
solution.  On  heating,  white  urate  of  copper  separates  out.  If  more 
copper  solution  is  added,  a  partial  reduction  of  the  cupric  oxide 
occurs,  owing  to  the  formation  of  allantoin. 

Denxigi!:s'  Test. — If  uric  acid  is  transformed  into  alloxan  by 
means  of  nitric  acid,  and  the  excess  of  acid  is  carefully  evaporated, 
a  blue  color  results  on  treating  the  residue  with  a  few  drops  of 
concentrated  sulphuric  acid  and  commercial  benzol  containing 
thiophen. 

Schiff's  Test. — If  a  piece  of  filter-paper  is  moistened  with  a 
solution  of  nitrate  of  silver,  and  a  drop  of  a  solution  of  uric  acid  in 
sodium  carbonate  is  added,  a  brownish-black  color  develops,  owing 
to  reduction  of  the  oxide  of  silver.  In  the  presence  of  only  0.002 
milligramme  of  uric  acid  a  yellow  color  is  obtained. 

Isolation  of  Uric  Acid. — Uric  acid  is  most  conveniently  prepared 
from  the  excrements  of  snakes,  in  Avhich,  as  has  been  stated,  it 
exists  in  the  form  of  the  quadriurate.  To  this  end,  the  material  is 
boiled  with  a  dilute  solution  of  sodium  hydrate  so  long  as  ammonia 
is  evolved,  when  carbon  dioxide  is  passed  through  the  solution  until 
the  alkaline  reaction  has  largely  disappeared.  The  acid  biurate  of 
sodium  which  separates  out  is  then  washed  with  cold  water  and 
dissolved  in  a  dilute  sodium  hydrate  solution.  On  adding  an 
excess  of  concentrated  hydrochloric  acid  the  uric  acid  crystallizes 
out  on  standing. 

From  human  urine  the  substance  can  be  obtained  by  adding  con- 
centrated hydrochloric  acid  in  the  proportion  of  50  :  1000,  and 
keeping  the  mixture  at  a  low  temperature  for  about  forty-eight 
hours.  The  crystals  which  then  separate  out  are  treated  with 
water,  dissolved  in  dihite  sodium  hydrate  solution,  decolorized  with 
animal  charcoal,  and  reprecipitated  with  hydrochloric  acid.  This 
method  was  formerly  employed  for  estimating  the  amount  of  uric 
acid  in  the  urine,  but  has  now  been  abandoned,  as  it  does  not  furnish 
reliable  results  and  in  its  place  the  method  of  Hopkins  or  of 
Ludwig-Saikowski  is  now  almost  exclusively  used  (see  below). 

Quantitative  Estimation. — HopKl^"s'  Method. — This  method 
furnislies  results  which  are  as  accurate  as  those  obtained  with  the 
older  method  of  Ludwig-Salkowski,  and  has,  above  all^  the  advan- 


THE   ORGANIC  CONSTITUENTS  OF  THE    URINE.  257 

tage  of  greater  simplicity.  It  is  based  upon  the  fact  that  uric  acid 
can  be  completely  precipitated  from  the  urine  by  the  addition  of  cer- 
tain ammonium  salts.  Insoluble  acid  ammonium  urate  thus  results, 
which  is  transformed  into  the  free  acid,  and  this  estimated  either 
gravimetrically  or  by  titration  with  a  solution  of  potassium  per- 
manganate of  known  strength.  According  to  Folin's  most  recent 
modification  of  the  original  method,  we  may  proceed  as   follows  : 

FoLix's  Method. — To  precipitate  the  uric  acid,  and  also  to  re- 
move the  small  amount  of  mucoid  substance  whicli  is  found  in 
every  urine,  the  following  reagent  is  employed  :  500  grammes  of 
ammonium  sulphate,  5  grammes  of  uranium  acetate,  and  60  c.c.  of  a 
10  per  cent,  solution  of  acetic  acid  are  dissolved  in  650  c.c.  of  water. 
The  resulting  solution  measures  about  1000  c.c.  Seventy-five  c.c.  of 
the  reagent  are  added  to  -300  c.c.  of  urine  in  a  flask  holding  500  c.c. 
After  standing  for  five  minutes  the  mixture  is  filtered  through  two 
folded  filters,  and  thus  freed  from  the  mucoid  body,  which  is  carried 
down  with  the  uranium  phosphate  in  acid  solution.  The  filtrate  is 
divided  into  two  portions  of  125  c.c.  each,  which  are  placed  in 
beakers  and  treated  with  5  c.c.  of  concentrated  ammonia.  After 
stirring  a  little  the  solutions  are  set  aside  until  the  next  day.  The 
supernatant  fluid  is  then  carefully  poured  off  through  a  filter 
(Schleicher  and  Schiill  No.  597) ;  the  precipitated  ammonium 
urate  is  collected  with  the  aid  of  a  small  amount  of  a  10  per 
cent,  solution  of  ammonium  sulphate  and  washed  with  the  same 
reagent.  Traces  of  chlorides  do  not  interfere  with  the  subsequent 
titration,  and  the  process  of  filtration  and  washing  can  be  completed 
in  from  twenty  to  thirty  minutes.  The  ammonium  urate  is  then 
washed  into  a  beaker,  after  ojiening  the  filter,  using  about  100  c.c. 
of  water.  Fifteen  c.c.  of  concentrated  sulphuric  acid  are  then  added 
and  the  solution  is  titrated  at  once  with  a  one-twentieth  normal  solu- 
tion of  potassium  permanganate.  Toward  the  end  of  the  titration 
Folin  suggests  to  add  the  permanganate  in  portions  of  two  drops  at 
a  time,  until  i\\e  first  trace  of  rose  is  apparent  throughout  the  entire 
fluid.  Each  cubic  centimeter  of  the  reagent  corresponds  to  0.00375 
gramme  of  uric  acid.  A  final  correction  of  0.003  gramme  for  every 
100  c.c.  of  urine  employed  is  necessary,  owing  to  the  slight  degree 
to  which  ammonium  urate  is  soluble. 

Ludwig-Salkowski  Method. — This  is  based  upon  the  forma- 
tion of  insoluble  magnesium  silver  urate  when  urine  is  treated  with 
ammoniacal  magnesia  mixture  and  subsequently  with  an  ammoniacal 
solution  of  silver  nitrate,  while  the  cldorides  remain  in  solution. 
The  double  salt  is  then  decomposed  and  the  uric  acid  obtained  as 
such,  or  the  amount  of  silver  is  ascertained  by  titration  and  the  cor- 
responding amount  of  uric  acid  calculated.  The  gravimetric  method 
is  probably  the  most  accurate.  The  titration  method  presupposes 
that  the  composition  of  magnesium-silver  urate  is  constant,  viz.,  that 
it  contains  one  molecule  of  uric  acid  for  every  atom  of  silver ;  Ijut 
this  has  not  been  definitely  established. 
17 


258  THE   URINE. 

For  clinical  purposes,  however,  tlic  titration  method  may  be  em- 
ployed, as  the  error  which  is  thus  involved  is  probably  only  slip:ht. 
Method. — Two  hundred  and  fifty  c.c.  of  urine,  which  should 
present  a  specific  gravity  approximating  1.020,  are  treated  with  50 
c.c.  of  ammouiacal  magnesia  mixture.  (This  is  prepared  by  dis- 
solvino-  100  grammes  of  magnesium  chloride  in  water,  adding  a  cold 
saturated  solution  of  ammonium  chloride  in  excess,  and  enough 
strong  ammonia  to  impart  a  decided  odor.  If  the  mixture  is  not 
clear,  more  of  the  ammonium  chloride  solution  is  added,  and  it  is 
finally  diluted  with  water  to  the  1000  c.c.  mark.)  After  filtering 
oif  the  phosphates,  which  are  precipitated  by  the  magnesium 
mixture,  240  c.c.  of  the  filtrate,  corresponding  to  200  c.c.  of  urine, 
are  treated  with  20  c.c.  of  an  ammoniacal  3  per  cent,  silver  nitrate 
solution.  In  this  manner  the  uric  acid,  as  also  the  xanthin  bases, 
are  precipitated  as  silver  salts,  and  are  allowed  to  settle.  A  few 
cubic  centimeters  of  the  clear  supernatant  fluid  are  then  examined 
to  ascertain  whether  a  sufficient  quantity  of  the  silver  solution  has 
been  added.  To  this  end,  it  is  only  necessary  to  acidify  with  con- 
centrated nitric  acid,  when  a  precipitate  of  silver  chloride  should 
occur.  If  this  does  not  happen,  the  specimen  is  again  rendered 
alkaline  with  ammonia,  poured  back,  and  the  entire  volume  treated 
with  a  little  more  of  the  silver  solution. 

The  gelatinous  precipitate  is  filtered  oif  with  the  aid  of  a 
suction-pump,  washed  free  from  chlorides  and  silver  with  weak 
ammonia-water,  and  transferred  to  a  beaker.  Twenty  c.c.  of  a 
solution  of  sodium  sulphide,  diluted  with  an  equal  volume  of  waier, 
are  brought  to  the  boiling-point,  and  then  immediately  added. 
(The  sulphide  solution  is  prepared  by  dissolving  10  grammes  of 
chemically  pure  sodium  hydrate  in  1000  c.c.  of  distilled  water;  one- 
half  of  this  volume  is  saturated  with  hydrogen  sulphide  and  mixed 
with  the  other  half.)  A  little  more  boiling  w^ater  is  added  to  the 
mixture,  which  is  kept  on  the  water-bath  for  a  while.  As  soon 
as  the  supernatant  fluid  is  perfectly  colorless  the  solution  is  allowed 
to  cool.  The  sulphide  of  silver  is  then  filtered  off  and  washed  with 
hot  water.  Filtrate  and  washings  are  then  acidified  with  hydro- 
chloric acid  and  evaporated  to  about  15  c.c. 

After  adding  a  little  more  hydrochloric  acid  the  solution  is 
allowed  to  stand  for  twenty-four  hours,  when  the  uric  acid  is 
filtered  oif,  washed  with  water,  then  with  alcohol,  and  dried  at 
115°  C.  For  every  10  c.c.  of  the  mother-liquor  and  water  used  in 
the  final  washing,  0.00048  gramme  is  finally  added  to  the  result, 
to  allow  for  the  trifling  amount  of  uric  acid  which  remains  in 
solution. 

Instead  of  decomposing  the  silver  compounds  as  described  with 
sodium  sulphide,  we  may  proceed  as  follows :  the  precipitate  is 
suspended  in  about  300  c.c.  of  acidulated  water  and  subjected  to  a 
current  of  hydrogen  sulphide  until  the  decomposition  is  complete ; 
on  subsequent  boiling  all  the  uric  acid  passes  into  solution,  and  can 


THE   ORGANIC  CONSTITUENTS  OF  THE   URINE.  259 

be  separated  from  the  precipitate  of  silver  sulphide  by  filtration. 
Filtrate  and  washing^s  are  then  further  treated  as  described. 
Albumin  and  sugar,  if  present,  must  in  either  case  be  removed. 

The   Xanthin   Bases. 

The  xanthin  bases  which  have  been  found  in  the  urine  of  man 
are  xanthin,  hypoxanthin,  guanin,  carnin,  paraxanthin,  heteroxan- 
thin,  episarcin,  and  under  certain  pathological  conditions  adenin. 
Their  amount,  however,  is  always  small,  and  normally  constitutes 
about  10  per  cent,  of  the  quantity  of  uric  acid,  viz.,  from  0.02  to 
0.06  gramme.  Of  this  amount,  from  0.02  to  0.03  gramme  is  repre- 
sented by  xanthin.  Hypoxanthin  and  guanin  probably  stand  next 
in  order,  while  paraxanthin  and  heteroxanthin  are  found  only  in 
traces.  From  10,000  liters  of  urine  Kriiger  and  Salomon  thus 
obtained  only  7.5  grammes  of  the  latter. 

Origin. — It  has  been  shown  that  the  xanthin  bases  are  derived 
from  the  nuclear  nucleins,  and  are  probably  formed  in  all  the  tissues 
of  the  body.  There  is  reason  to  suppose,  moreover,  that  a  certain 
fraction  is  referable  to  ingested  nucleins.  Under  normal  conditions 
the  greater  portion  of  the  xanthin  bases  is  then,  no  doubt,  oxidized 
to  uric  acid,  but  a  variable  fraction  escapes  as  such.  To  a  certain 
extent  the  oxidation  to  uric  acid  occurs  in  the  liver,  but,  as  I  have 
shown,  this  takes  place  also  in  other  organs  of  the  body,  as  both 
xanthin  bases  and  uric  acid  are  obtained  in  transfusion  experiments. 
At  the  same  time  it  was  noted  that  the  relative  amount  of  the  two 
was  largely  influenced  by  the  degree  of  oxygenation  of  the  blood,  so 
that  xanthin  bases  only  were  obtained  if  venous  blood  was  used, 
while  both  were  found  when  arterial  blood  was  employed.  We 
can  thus  understand  that,  as  a  general  rule,  at  least  a  certain  rela- 
tion exists  in  the  elimination  of  uric  acid  and  the  xanthin  bases. 
A  diminished  elimination  of  the  latter  is  thus  quite  frequently 
associated  with  a  corresponding  increase  of  the  former,  or  vice  versa, 
and  both  may,  of  course,  be  increased  or  diminished  together.  The 
most  notable  increase  in  their  elimination  is  observed  in  leukaemia, 
and  here  adenin  also  appears  in  the  urine. 

Theobromin  (dimethyl-xanthin)  and  cafFein  (trimetfiyl-xanthin) 
are  partly  eliminated  in  the  urine  as  such,  and  partly  appear  as  a 
methyl-xanthin  which  is  apparently  identical  with  heteroxanthin. 

Xanthin  has  once  been  found  in  crystalline  form  in  a  urinary 
sediment,  and  has  in  several  instances  been  encountered  in  vesical 
calculi. 

As  the  isolation  of  the  individual  substances  from  the  urine  in 
amounts  sufficient  for  purposes  of  study  is  a  very  complicated  process, 
and  requires  facilities  which  are  not  generally  found  in  university 
laboratories,  it  will  suffice  at  this  place  to  describe  a  method  by 
which  they  can  be  collectively  estimated.  For  a  consideration  of 
the  chemical  properties  of  the  more  important  members  of  the  group 


260  THE   URINE. 

and  their  isolation  from  other  sources,  as  also  of  their  relation  to 
uric  acid  and  the  nucleinic  bases,  the  reader  is  referred  to  other 
sections. 

Quantitative  Estimation. — The  xanthin  bases  are  best  isolated 
from  the  urine  according  to  Salkowski's  method,  which  is  based 
upon  their  precipitation  as  silver  compounds,  together  with  uric  acid, 
the  separation  of  the  latter,  and  the  determination  of  the  amount  of 
silver  in' combination  with  the  bases.  To  this  end,  600  c.c.  of 
urine  are  first  treated,  as  described  in  the  quantitative  estimation 
of  uric  acid,  according  to  Ludwig-Salkowski.  The  final  filtrate 
after  removal  of  the  uric  acid,  together  with  the  washings,  is  then 
treated  with  ammoniacal  silver  solution  and  the  xanthin  bases  thus 
reprecipitated.  The  precipitate  is  collected  on  a  small  filter,  washed 
with  water,  dried,  and  incinerated.  The  ash  is  dissolved  in  dilute 
nitric  acid,  and  the  silver  estimated  by  titrating  with  a  solution  of 
potassium  sulphocyanide  of  known  strength,  using  ammonio-ferric 
alum  as  an  indicator.  As  it  has  been  ascertained  that  in  an  equal 
mixture  of  the  silver  compounds  of  xanthin,  hypoxanthin,  guanin, 
etc.,  one  atom  of  silver  represents  0.277  gramme  of  nitrogen  or 
0.7381  gramme  of  the  bases,  1  c.c.  of  the  sul])hocyanide  solution, 
that  is  commonly  used  in  the  estimation  of  the  chlorides  of  the 
urine  (page  237),  will  correspond  to  0.002  gramme  of  nitrogen  or 
0.00542  gramme  of  the  bases. 

The  method  of  Krliger  and  WulflP,  which  was  greatly  in  vogue  a 
few  years  ago,  has  been  abandoned,  as  the  values  thus  obtained  were 
too  high.  According  to  this  method,  the  alloxuric  bodies,  viz.,  uric 
acid  and  xanthin  bases,  were  first  estimated  by  precipitating  with 
copper  sulphate  and  sodium  bisulphite  and  determining  the  amount 
of  nitrogen  in  the  precipitate.  In  a  second  portion  of  the  urine  the 
uric  acid  Avas  then  estimated  and  the  corresponding  amount  of 
nitroo-en  deducted  from  the  first  result.  The  difference  was  referred 
to  the  xanthin  bases. 

Oxalic  Acid  and  Oxaluric  Acid. 

Of  the  origin  of  oxalic  acid  and  oxaluric  acid,  both  of  which  may 
oe  regarded  as  normal  constituents  of  the  urine,  but  little  is  known. 
The  former  is  supposedly  present  as  a  calcium  salt,  which  is  held 
in  solution  owing  to  the  presence  of  diacid  sodium  phosphate,  but 
readily  separates  out  on  standing  and  is  then  frequently  encountered 
in  urinary  sediments.  Here  it  generally  occurs  in  the  very  charac- 
teristic envelope  or  dumb-bell  forms,  and  can  be  readily  distin- 
guished from  other  constituents  by  its  insolubility  in  acetic  acid, 
and  its  solubility  in  hydrochloric  acid.  Its  amount  normally  varies 
between  0.02  and  0.05  gramme.  Oxaluric  acid,  on  the  other  hand, 
exists  in  the  urine  as  an  ammonium  salt  and  is  not  found  in  sedi- 
ments.    Its  amount  is  even  smaller  than  that  of  oxalic  acid. 

As  many  articles  of  food,  such    as   asparagus,  spinach,  grapes, 


THE  ORGANIC  CONSTITUENTS   OF  THE   URINE. 


261 


apples,  etc.,  contain  oxalic  acid  in  not  inconsiderable  amounts,  it  is 
supposed  that  a  certain  fraction  of  the  oxalic  acid  of  the  urine  is 
referable  to  this  source.  We  find,  as  a  matter  of  fact,  that  in  the 
asparagus  season  larger  amounts  are  eliminated  than  at  any  other 
time  of  the  year.  But  it  has  also  been  noted  that  oxalic  acid  does 
not  disappear  from  the  urine  when  the  diet  consists  exclusively  of 
albumins  and  fats,  and  that  during  starvation  also  oxalic  acid  can 
still  be  found.  We  are  consequently  forced  to  the  conclusion  that 
a  certain  amount  of  the  substance  must  originate  in  the  tissues  of 
the  body,  and  there  is  a  growing  belief  that  the  albumins  are  here 
its  ultimate  source.  We  know  indeed  that  oxaluric  acid  is  closely 
related  to  uric  acid,  and  it  in  turn  can  be  decomposed  into  urea  and 
oxalic  acid,  as  is  shown  by  the  equations : 


(1)  CsH.NA  +  O  +  H,0 

Uric  acid. 


CO  —  NH. 

I  \ 

(2)  CO  >C0+0 

I  / 

CO  —  NH/ 
Alloxan. 


CO  —  NH^ 

I 

CO 

CO  —  NH 

Alloxan. 


/ 


€0  +  CO(NH2)2 


CO  —  NH 


CO  —  NH-^ 

Parabanic  acid. 


^CO  +  CO2 


CO NH. 

(3)    I    .  >C0 

CO NH/ 

Parabanic 
acid. 


H,0 


CO- 


NHs 


COOH.NH2 

Oxaluric  acid. 


)C0 


CO NH. 

(4)    I  >C0  +  H.,0 

COOH.NH./ 
Oxaluric  acid. 


CO- 
CO- 


-OH 
-OH 


Oxalic  acid. 


/NH, 

C0< 

\nh. 

Urea. 


As  oxalic  acid  on  further  oxidation  is  decomposed  into  water  and 
carbon  dioxide,  it  would  thus  appear  that  both  oxaluric  acid  and 
oxalic  acid  may  be  regarded  as  complete  oxidation-products  of  uric 
acid.  We  find,  as  a  matter  of  fact,  that  oxalic  acid  is  increased  in 
various  diseases  in  which  the  oxidation-processes  are  manifestly 
impaired,  such  as  diabetes  mellitus,  various  diseases  of  the  circulatory 
apparatus  when  associated  with  deficient  oxygenation  of  the  blood, 
in  obesity,  etc.  I  have  frequently  observed,  moreover,  that  an 
increased  elimination  of  oxalic  acid  is  associated  with  an  increased 
excretion  of  uric  acid  in  young,  more  or  less  anaemic  individuals  of 
a  neurotic  type.  Whether  or  not  oxalic  acid  may  further  be  derived 
from  carbohydrates  is  as  yet  unknown,  but  is  rather  improbable. 
Aside  from  its  occurrence  in  solution  and  in  urinary  sediments,  oxalic 
acid  is  also  not  infrequently  found  to  constitute  the  greater  portion 
of  renal  and  vesical  calculi. 

Quantitative    Estimation    of   Oxalic   Acid. — Dunlop's    Method 


262  THE   URINE. 

(modified  by  Baldwin). — Five  hundred  c.c.  of  a  well-mixed  specimen 
of  the  twenty-four  hours'  urine  are  treated  with  150  c.c.  of  ov'er  90 
per  cent,  alcohol,  to  precipitate  the  oxalate  of  calcium.  After  forty- 
eight  hours  the  crystals  are  collected  on  a  filter,  thoroughly  washed 
with  hot  and  cold  water  and  with  dilute  acetic  acid  (1  per  cent.).  The 
filter  is  placed  in  a  small  beaker  and  soaked  in  a  small  amount  of 
dilute  hydrochloric  acid.  It  is  then  washed  with  hot  water  until 
there  is  no  further  acid  reaction.  The  washings  are  filtered  and 
evaporated  to  about  20  c.c.  A  very  little  calcium  chloride  solution 
is  added  to  insure  an  excess  of  calcium.  The  hydrochloric  acid  is 
neutralized  Avith  ammonia  and  the  solution  then  rendered  slightly 
acid  with  acetic  acid.  Strong  alcohol  is  now  added  to  the  amount 
of  50  per  cent,  of  the  volume  of  the  fluid  and  the  solution  set  aside 
for  forty-eight  hours.  The  sediment  is  collected  on  a  filter  that  is 
free  from  ash,  and  washed  with  cold  water  and  dilute  acetic  acid  until 
free  from  chlorides.  (Hot  water  should  here  be  avoided,  as  it  carries 
the  finely  divided  precipitate  through  the  pores  of  the  filter.)  The 
filter  is  incinerated  over  a  Bunsen  burner,  and  afterward  heated  in 
the  blowpipe-flame.  The  residue  is  allowed  to  cool  over  sulphuric 
acid  and  weio^hed.  The  ash  is  calcium  oxide,  each  gramme  of  which 
corresponds  to  1.6  grammes  of  oxalic  acid. 

The  urine  in  every  case  should  l)e  thymolized  as  soon  as  possible, 
to  prevent  fermentation  and  the  precipitation  of  phosphates.  If  the 
specimen  is  alkaline,  it  is  rendered  slightly  acid  with  acetic  acid. 

The  method  is  applicable  in  the  case  of  human  urine,  but  in 
that  of  dogs  with  a  high  specific  gravity  it  is  very  difficult  to  remove 
the  phosphates.  In  such  an  event  Salkowski's  method  is  best 
employed. 

Salkowski's  Method. — If  the  urine  is  concentrated  (sp.  gr. 
1.040-1.050),  it  is  treated  with  20  c.c.  of  hydrochloric  acid  (sp.  gr. 
1.12)  for  200-250  c.c,  and  extracted  in  a  separating  funnel  three 
times  with  alcoholic  ether  (5-10  per  cent,  alcohol).  The  ethereal 
extract  is  filtered  through  a  dry  filter,  the  ether  is  distilled  off",  the 
remaining  fluid  evaporated  to  20  c.c,  and  filtered  on  cooling.  The 
filtrate  is  rendered  alkaline  with  ammonia,  and  is  then  treated  with 
1—2  c.c.  of  a  10  per  cent,  solution  of  calcium  chloride  and  acetic 
acid.     The  process  is  continued  as  described. 

AVith  human  urine  larger  quantities,  such  as  500  cc,  are  em- 
ployed, which  are  first  concentrated  to  about  one-third  of  their 
original  volume. 

AUantoin. 

Allantoin  is  a  normal  constituent  of  the  urine  of  man,  as  also 
of  various  animals,  but  is  usually  present  only  in  traces  in  the 
adult,  while  during  the  first  weeks  of  life  it  is  more  abundant. 
Larger  amounts  also  occur  during  pregnancy.  Aside  from  the 
urine,  it  is  found  in  the  amniotic  fluid  and  in  the  allantoic  fluid  of 
cows.     Like  oxaluric  acid,  it  is  an  oxidation-product  of  uric  acid, 


THE  OBOANIC  CONSTITUENTS  OF  THE    URINE.  263 

and  Salkowski  demonstrated  that  an  increased  elimination  occurred 
in  dogs  when  uric  acid  was  ingested.  An  artificial  increase  is 
similarly  observed  in  poisoning  with  hydrazin,  and  we  can  readily 
understand  that  in  consequence  of  the  degenerative  changes  which 
are  thus  produced  in  the  liver  the  oxidation-processes  are  accordingly 
diminished,  and  allantoin  results. 

The  formation  of  allantoin  from  uric  acid  may  be  represented  by 
the  equation  : 

.NH.CH.NH.CO.NH, 


C5H4N,03  +  H^O  +  O  =  CO'/"  '  +  CO2 

^NH.CO 

Uric  acid.  Allantoin. 


It  is  a  glyoxyl-diureid,  and  may  be  produced  artificially  by 
heating  glyoxylic  acid  and  urea  together  at  a  temperature  of  100°  C 
The  reaction  then  takes  place  according  to  the  equation  : 

/NHj         COH  /NHj,                  /NH.CH.NH.CO.KH2 

C0<            +     I             +  C0<             =    C0(                                       +    2H,0 

^NHj         COOH  \NH2                  \NH.C0 

Urea.        Glyoxylic  Urea.                                      Allantoin. 
acid. 

The  substance  crystallizes  in  colorless  rhombic  prisms,  which  are 
frequently  grouped  in  the  form  of  stars  and  rosettes.  It  is  soluble 
with  some  difficulty  in  cold  water,  more  readily  in  hot  water  and 
hot  alcohol,  while  in  cold  alcohol  and  ether  it  is  insoluble.  On  pro- 
longed boiling,  it  reduces  Fehling's  solution,  and  it  is  owing  to  the 
formation  of  this  substance,  as  already  indicated,  that  uric  acid  can 
reduce  cupric  oxide  when  boiled  in  alkaline  solution  (see  page  256). 
With  silver  nitrate  allantoin  forms  a  crystalline  precipitate  of  allan- 
toin silver,  if  ammonia  is  cautiously  added  to  the  solution,  but  it  is 
soluble  in  an  excess  of  the  alkalies. 

Isolation. — Allantoin  is  most  conveniently  obtained  from  the 
urine  of  calves.  To  this  end,  the  urine  is  evaporated  on  a  water- 
bath  to  a  thick  syrup  and  allowed  to  stand  for  several  days  in  the 
cold.  The  crystalline  constituents  which  have  formed  are  then 
separated  mechanically  from  the  mother-liquor  and  the  gelatinous 
material  which  is  present,  and  dissolved  in  a  small  amount  of  hot 
water.  The  solution  is  decolorized  with  animal  charcoal,  filtered 
while  hot,  and  the  filtrate  acidified  slightly  with  hydrochloric  acid. 
On  standing,  the  allantoin  separates  out. 

Kreatinin. 

The  kreatinin  which  is  found  in  the  urine  of  man  and  all 
mammals  is  in  all  probability  derived  from  the  kreatin  of  the 
muscle-tissue,  and  is  thus  in  part  referable  to  the  ingested  animal 
food,  and  in  part  to  the  wear  and  tear  which  constantly  goes  on  in 
the  muscular  structures  of  the  body.  The  latter  source,  as  com- 
pared with  the  former,  is  normally,  however,  of  secondary  impor- 
tance, as  the  greater  amount  of   kreatin    which   originates    in    the 


264  THE   URINE. 

muscles  is,  in  health  at  least,  transformed  into  urea.  We  accord- 
ingly find  that  the  elimination  of  kreatin  is  much  reduced  when  the 
animal  is  placed  on  a  diet  of  milk  exclusively,  while  it  is  increased 
if  a  liberal  amount  of  meat  is  ingested,  or  kreatin  is  administered 
as  such.  On  the  other  hand,  we  observe  that  muscular  exercise  in 
itself  does  not  call  forth  an  increased  excretion,  although  this  has 
recently  been  denied.  Under  pathological  conditions,  however,  in 
which  an  increased  destruction  of  the  albuminous  constituents  of  the 
body  occurs,  abnormally  large  quantities  may  be  found  in  the  urine, 
although  the  patient  receives  food  which  is  free  from  kreatin.  In 
such  cases  we  may  assume  that  the  muscles  have  lost  to  a  greater  or 
less  degree  the  power  of  transforming  kreatin  into  urea.  Kreatin 
itself  has  thus  far  not  been  found  in  perfectly  fresh  urine,  but  is 
formed  from  the  kreatinin  on  the  occurrence  of  bacterial  decomposi- 
tion. The  transformation  of  kreatin  into  its  anhydride  supposedly 
occurs  in  the  kidneys,  and  we  accordingly  find  that  in  extensive 
disease  of  these  organs  the  elimination  of  kreatinin  is  diminished. 

Of  late,  it  has  been  claimed  that  the  kreatinin  which  is  found  in 
muscle-tissue  is  not  identical  with  the  urinary  kreatinin,  and  it  has 
even  been  suggested  that  the  source  of  the  latter  may  have  to  be 
sought  in  other  organs  of  the  body,  and  notably  in  the  thyroid 
gland,  in  which  kreatinin  has  been  found  in  considerable  amount. 
It  is,  of  course,  possible  that  the  substance  may  be  formed  in  other 
organs  as  well,  but  the  evidence  is  conclusive  that  the  urinary  form 
is  largely  derived  from  muscle-tissue.  But  even  supposing  that 
future  researches  should  show  that  the  two  bodies  are  isomeric,  but 
not  identical,  we  could  even  then  imagine  that  both  originate  from 
the  same  substance. 

The  amount  of  kreatinin  which  is  normally  found  in  the  urine  of 
man  is  approximately  1  gramme,  but  varies,  of  course,  with  the 
character  of  the  food. 

Properties. — Kreatinin  crystallizes  in  colorless,  highly  refractive 
monoclinic  prisms,  and  is  quite  soluble  in  hot  and  cold  water,  less 
readily  so  in  alcohol,  and  is  insoluble  in  ether.  In  aqueous  solution 
it  is  gradually  transformed  into  kreatin.  The  same  transformation 
results  more  rapidly  when  the  substance  is  heated  in  alkaline  solu- 
tion. It  combines  with  acids  and  various  salts  to  form  crystalline 
compounds,  some  of  which  are  characteristic.  This  is  true  especially 
of  the  chlon^zincate — {QJi.^'N^O^,,.7jnC\., — which  results  when  a  con- 
centrated alcoholic  solution  of  kreatinin  is  treated  with  a  solution 
of  zinc  chloride,  wdiich  should  be  as  little  acid  as  possible.  The 
crystalline  form  of  this  compound  de])ends  very  much  ujwn  its 
purity.  As  first  obtained  from  the  urine,  it  occurs  in  the  form  of 
varicose  conglomerations,  which  usually  adhere  firmly  to  the  walls 
of  the  vessel.  In  pure  form  it  crystallizes  in  fine  needles,  which 
are  commonly  grouped  in  sheaves  and  stars.  The  salt  is  almost 
insoluble  in  alcohol,  and  nearly  so  in  water,  while  free  mineral 
acids  dissolve  it. 


THE   ORGANIC  CONSTITUENTS  OF  THE   URINE.  265 

Kreatinin  is  further  precipitated  by  mercuric  nitrate,  notably  in 
the  presence  of  sodium  carbonate,  by  silver  nitrate,  platinum 
chloride,  stannous  chloride,  mercuric  chloride,  picric  acid,  phospho- 
tungstic  acid,  etc.  With  all  these  substances  it  forms  well-charac- 
terized crystalline  salts.  In  alkaline  solution  it  reduces  cupric 
hydroxide,  and  it  is  for  this  reason  that  urines  which  contain  much 
kreatinin  but  no  sugar  give  a  positive  reaction  with  Fehling's 
solution.  The  separation  of  the  cupric  oxide,  however,  only  occurs 
on  prolonged  boiling.  An  alkaline  solution  of  bismuth,  on  the 
other  hand,  is  not  aifected. 

Tests. — Weyl's  Test. — A  few  cubic  centimeters  of  urine  are 
treated  with  a  few  drops  of  a  freshly  prepared,  very  dilute  solution 
of  sodium  nitroprusside,  and  then  drop  by  drop  with  a  solution 
of  sodium  hydrate,  when  in  the  presence  of  kreatinin  a  ruby-red 
color  develops,  which  is  especially  pronounced  in  the  lower  portion 
of  the  tube.  After  a  few  minutes  the  color  disappears.  If  now 
acetic  acid  is  added  in  excess  and  the  mixture  heated,  it  assumes  a 
greenish  color,  then  turns  blue,  and  on  standing  deposits  a  sediment 
of  Prussian  blue.  Acetone  and  diacetic  acid  give  a  similar  reaction 
if  ammonia  is  added  instead  of  sodium  hydrate ;  but  with  kreatinin 
no  red  color  is  thus  obtained. 

Jaffe's  Test. — If  a  few  cubic  centimeters  of  urine  are  treated 
with  a  dilute  aqueous  solution  of  picric  acid,  the  corresponding 
compound  of  kreatinin  is  precipitated.  On  adding  a  few  drops  of 
a  dilute  solution  of  sodium  hydrate  a  red  color  develops,  which  per- 
sists for  several  hours,  and  is  changed  to  yellow  upon  the  addition 
of  an  acid.  With  glucose  a  red  color  also  is  obtained,  but  only  on 
heating,  while  the  reaction  in  the  case  of  kreatinin  takes  place  only 
at  ordinary  temperatures.     Acetone  gives  a  reddish-orange  color. 

Synthesis  of  Kreatinin. — Kreatinin  can  be  formed  synthetically 
from  methyl-glycocoU  and  cyanamide.  Kreatin  is  first  produced, 
and  then  yields  the  anhydride,  as  shown  in  the  equations  : 

/NH 

(1)  N=C  —  NH,  +  NH(CH3)CH2.COOH  =  NH  :  C< 

\N(CH,).CH2.C00H 
Cyanamide.  Methyl-glycocoll.  Kreatin. 

/NH,  /NH (X> 

(2)  NH=C<  =NH:0(  |       +  H,0 

\N(CH0.CH,.COOH  \N(CH3).CH, 

Kreatin.  Kreatinin. 

Isolation  and  Quantitative  Estimation. — The  quantitative  estima- 
tion of  kreatinin  in  the  urine  is  based  upon  the  formation  of  the 
chlorozincate,  which  is  almost  insoluble  in  alcohol :  240  c.c.  of 
urine,  which  should  be  free  from  albumin  and  sugar,  are  rendered 
alkaline  with  milk  of  lime,  and  treated  with  a  solution  of  calcium 
chloride  so  long  as  a  precipitate  forms.  Water  is  then  added  to  the 
300  c.c.  mark.  After  standing  for  a  while  the  phosphates  are 
filtered  off.     The  precipitate  is  washed  with  a  little  water.     Filtrate 


266  THE   URINE. 

and  washings  are  rendered  slightly  acid  with  acetic  acid,  and  are 
then  evaporated  to  a  syrup.  This  is  stirred  while  still  warm  with 
about  25  to  30  c.c.  of  absolute  alcohol,  transferred  to  a  glass- 
stoppered  flask,  and  diluted  with  absolute  alcohol  to  100  c.c.  After 
twenty-four  hours  the  mixture  is  filtered.  The  filtrate  is  treated 
with  a  small  amount  of  sodium  acetate  in  solution,  and  is  con- 
centrated to  about  50  c.c.  To  this  is  added  0.5  c.c.  of  a  concentrated 
alcoholic  solution  of  zinc  chloride,  which  is  prepared  by  dissolving 
a  small  amount  of  the  salt  in  80  per  cent,  alcohol,  and  diluting  with 
95  per  cent,  alcohol  to  a  specific  gravity  of  1.2.  The  mixture  is 
then  well  stirred  and  set  aside  in  a  cool  place  for  several  days. 
The  crystals  of  the  chlorozincate  of  kreatinin  are  now  collected 
on  a  previously  weighed  filter  and  washed  with  alcohol  until  free 
from  chlorides,  when  they  are  dried  at  100°  C.  and  weighed. 
The  corresponding  amount  of  kreatinin  is  ascertained  by  multiply- 
ing the  weight  by  0.6243.  The  material  which  is  thus  obtained 
is,  however,  always  impure,  owing  to  an  admixture  of  various  pig- 
ments and  traces  of  chlorides.  If  greater  accuracy  is  required,  it  is 
hence  necessary  to  determine  the  amount  of  zinc  in  the  crystals, 
which  may  be  done  as  follows :  the  material  is  covered  with  a 
little  nitric  acid  ;  the  solution  is  evaporated,  the  residue  incinerated, 
extracted  with  water,  and  the  aqueous  solution  evaporated,  the 
residue  ignited,  and  finally  weighed.  The  zinc  is  thus  obtained  as 
oxide,  from  which  the  corresponding  amount  of  kreatinin  is  cal- 
culated by  multiplying  by  2.7790. 

To  isolate  the  kreatinin  from  the  chlorozincate,  the  latter  is  dis- 
solved in  a  small  amount  of  hot  water  and  lioiled  for  ten  minutes 
with  well-washed  plumbic  hydrate.  After  filtering  off  the  insoluble 
oxide  of  zinc  and  the  chloride  of  lead  the  filtrate  is  evaporated  to 
dryness  and  extracted  with  cold  absolute  alcohol.  This  takes  up 
the  kreatinin,  while  a  small  amount  of  kreatin  formed  during  the 
process  of  boiling  remains.  On  evajjoration  the  kreatinin  is  obtained 
in  crystalline  form,  and  can  be  further  purified  by  recrystallization 
from  water. 

THE  AROMATIC  CONSTITUENTS  OF  THE  URINE. 

It  has  been  pointed  out  in  a  preceding  chapter  that  during  the 
process  of  intestinal  putrefaction  various  aromatic  bodies  are  formed 
from  the  albumins  of  the  food  or  their  products  of  digestion,  and 
are  then  absorbed  and  eliminated  in  the  urine,  either  as  such  or  in 
combination  with  sulphuric  acid,  glucuronic  acid,  or  glycocoll. 
Some  of  these  bodies,  such  as  indol  and  skatol,  may  be  regarded  as 
specific  products  of  putrefaction  ;  while  others  or  closely  related 
substances  occur  preformed  also  in  many  articles  of  food.  AVe  con- 
sequently recognize  two  sources  of  the  aromatic  bodies  which  are 
found  in  the  urine,  viz.,  the  aromatic  bodies  which  epter  into  the 
composition  of  our  diet  as  such,  and  those  which  result  from  the 


THE  AROMATIC  CONSTITUENTS  OF  THE   URINE.         267 

destruction  of  albumins  through  the  activity  of  njicro-organisms. 
Under  certain  pathological  conditions,  further,  substances  of  this 
character  may  also  be  formed  in  the  body  proper,  owing  to  degenera- 
tive changes,  which  may  or  may  not  be  the  result  of  bacterial  action. 
Under  normal  conditions,  however,  this  source  scarcely  enters  into 
consideration. 

The  Conjugate  Sulphates. 

Paracresol,  phenol,  hydroquinon,  pyrocatechin,  indol,  and  skatol 
are  largely  eliminated  in  the  urine  in  combination  with  sulphuric 
acid  as  sodium  or  potassium  salts.  But  while  paracresol  and  its 
derivatives  combine  with  sulphuric  acid  directly,  indol  and  skatol 
are  previously  oxidized  to  indoxyl  and  skatoxyl,  as  has  been  shown. 
Conjointly  the  resulting  compounds  are  spoken  of  as  the  conjugate 
or  ethereal  sulphates  of  the  urine.  Their  daily  excretion  in  man 
corresponds  to  about  one-tenth  of  the  mineral  sulphates,  viz.,  from 
0.094  to  0.620  gramme,  under  normal  conditions.  Increased 
amounts  are  observed  when  from  whatever  cause  the  intestinal 
putrefaction  is  increased.  It  is  to  be  noted,  however,  that  the  ratio 
between  the  individual  substances  is  even  normally  not  constant, 
and  it  seems  that  the  relative  pre})onderance  of  the  one  over  the 
other  is  primarily  referable  to  the  extent  to  which  individual  groups 
of  micro-organisms  are  active.  In  some  instances  we  may  thus  find 
that  the  increase  in  the  conjugate  sulphates  is  referable  to  an  increased 
production  of  indol  and  skatol,  while  in  others  phenols  are  largely 
formed. 

Aside  from  an  increase  in  the  degree  of  intestinal  putrefaction, 
larger  amounts  of  the  conjugate  sulphates  may  also  be  observed  if 
putrefactive  processes  are  taking  place  within  the  body  proper,  pro- 
viding that  active  resorption  occurs  from  the  diseased  area.  An 
increased  elimination  is  also  noted  when  any  of  the  aromatic  sub- 
stances mentioned  are  ingested  as  such  or  otherwise  introduced  into 
the  circulation  from  without.  A  notable  increase  is  thus  observed 
in  poisoning  with  carbolic  acid  or  its  congeners,  and  is  then,  of 
course,  principally  owing  to  an  increased  formation  of  phenol  sul- 
phates. The  ingestion  of  ortho-nitro-phenyl-propiolic  acid,  which 
is  reduced  to  indoxyl  within  the  body,  similarly  leads  to  an  increased 
elimination  of  indoxyl  sulphate. 

The  synthesis  of  the  various  conjugate  sulphates  is  largely 
effected  in  the  liver,  but  may  also  occur  in  other  organs  of  the 
body,  such  as  the  kidneys  and  lungs.  In  some  experiments  posi- 
tive results  were  also  obtained  in  the  case  of  the  muscle-tissue  and 
the  intestinal  wall,  but  tiiese  organs  play  only  a  secondary  role  as 
compared  to  the  liver.  Their  quantitative  estimation  in  toto  has 
been  described. 

The  Phenols. — Of  the  phenols  which  occur  in  the  urine,  para- 
cresol is  the  most  abundant;  next  in  order  comes  phenol,  while 
pyrocatechin  and  hydroquinon  are  found  only  in  traces.     Besides 


268  THE   URINE. 

paracresol,  the  normal  urine  of  man  is  said  also  to  contain  minute 
amounts  of  mcta-  and  ortho-cresols.  The  total  elimination  of 
cresols  and  phenols,  however,  noniially  corresponds  to  only  about 
0.03  oramme    in    the    twenty-four    hours. 

Urine  whieli  contains  much  hydroquinon  or  pyrocatecliin  gradually 
assumes  a  dark-brown  color  on  standing  if  the  reaction  is  alkaline, 
and  it  is  noted  that  this  change  in  color  begins  in  the  upper  layers 
and  gradually  extends  downward.  Ultimately  the  urine  becomes 
almost  black.  This  change  is  referable  to  oxidation  of  the  dioxy- 
benzols,  but  of  the  resulting  compounds  nothing  is  known.  Urines 
of  this  character  are  mostly  observed  in  poisoning  with  carbolic  acid, 
following  the  administration  of  benzol,  of  phenol  sulphates,  etc. 

To  demonstrate  the  presence  of  phenol  or  of  paracresol,  1000 
c.c.  of  urine  are  treated  with  70  c.c.  of  concentrated  hydrochloric 
acid,  and  distilled  until  about  one-fourth  of  the  total  amount 
has  passed  over.  The  conjugate  sulphates  are  thus  decomposed, 
and  phenol  and  cresol  are  found  in  the  distillate.  Their  presence 
can  here  be  demonstrated  by  testing  with  Millon's  reagent  or  by 
adding  bromine-water,  when  tribromophenol  crystallizes  out  on 
standing.  If  much  phenol  is  present,  it  may  further  be  possible 
to  obtain  a  positive  reaction  with  ferric  chloride  solution  if  this  is 
added  drop  by  drop  in  very  dilute  solution.  Hydroquinon  and 
pyrocatecliin  remain  in  the  acid  sokition.  To  demonstrate  their  pres- 
ence, the  solution  is  evaporated  to  about  100  c.c,  and  on  cooling  is 
extracted  with  an  equal  volume  of  ether.  Hydroquinon  and  pyro- 
catechin  together  with  the  aromatic  oxy-acids  are  thus  removed. 
On  adding  a  dilute  solution  of  sodium  carbonate  to  the  ethereal  solu- 
tion the  aromatic  oxy-acids  are  transformed  into  the  corresponding 
sodium  salts.  The  ethereal  extract,  which  now  contains  only  the 
dioxy-benzols,  is  evaporated  to  dryness  ;  the  residue  is  dissolved  in 
a  little  water  and  examined  as  follows  :  one  portion  is  treated  drop 
by  drop  with  a  dilute  solution  of  ferric  chloride,  when  in  the  pres- 
ence of  pyrocatecliin  a  green  color  develops,  which  turns  to  violet 
upon  the  addition  of  a  small  amount  of  tartaric  acid  and  ammonia. 
The  remainder  of  the  solution  is  precipitated  with  lead  acetate  and 
filtered.  The  filtrate  contains  the  hydroquinon,  while  the  pyro- 
catecliin is  in  the  precipitate.  The  hydroquinon  can  then  be  isolated 
by  acidifying  and  extracting  with  ether,  when  the  sul)stance  crys- 
tallizes out  on  evaporation.  It  is  dissolved  in  a  little  water,  and 
treated  drop  by  drop  with  the  dilute  iron  solution.  C^uinon, 
CgH^Oj,  results,  and  may  be  recognized  by  its  penetrating  odor. 

Quantitative  Estimation. — Method  of  Kossler  and  Penny, 
MODIFIED  BY  Neuberg. — This  method  is  based  upon  the  precipi- 
tation of  phenol  and  paracresol,  by  means  of  iodine,  as  tri-iodo- 
plienol.  From  the  amount  of  iodine  which  is  thus  used  the  corre- 
sponding amount  of  monoxy-benzols  can  be  calculated,  and  is 
expressed  either  in  terms  of  phenol  or  cresol,  as  the  method  does 
not  indicate  the  separate  amount  of  the  individual  bodies  that  are 


THE  AROMATIC  CONSTITUENTS  OF  THE   URINE.         269 

present.     The  reaction  whi(!li  takes  place  may  be  represented  by 
the  equation  : 

CeHj.OH  +  61  =  CeH^JjOH  +  3HI. 

Five  hundred  c.c.  of  urine  are  rendered  feebly  alkaline  and 
evaporated  to  about  100  c.c.  Any  acetone  which  may  have  been 
present  is  thus  removed.  The  residual  fluid  is  acidified  with  sul- 
phuric acid,  so  as  to  contain  5  per  cent,  of  the  original  volume,  and 
is  then  repeatedly  distilled.  The  individual  portions  thus  obtained 
are  shaken  with  calcium  carbonate  until  the  acid  reaction  has  dis- 
a})peared,  so  as  to  remove  any  nitrous  or  formic  acid  that  may  be 
present.  The  fluid  is  now  again  distilled,  and  the  distillate  treated 
with  a  solution  of  1  gramme  of  caustic  soda  and  6  grammes  of  lead 
acetate  in  substance.  The  mixture  is  kept  on  a  boiling  water-bath 
for  about  fifteen  minutes.  A  portion  of  the  lead  oxide  is  thus  dis- 
solved by  the  phenol  to  form  basic  plienolatcs,  while  any  aldehydes 
or  ketones  that  may  have  been  formed  from  the  small  amount  of 
carbohydrates  that  are  present  in  every  urine  escape.  To  remove 
these  entirely,  the  mixture  is  heated  over  a  free  flame  connected  with 
a  condenser  until  a  few  cubic  centimeters  of  the  distillate  no  longer 
reduce  an  alkaline  solution  of  silver  nitrate.  After  five  minutes 
this  point  is  usually  reached.  The  fluid  is  then  acidified  with  sul- 
phuric acid  as  before,  and  is  distilled,  water  being  added  from  time 
to  time.  The  distillate  is  placed  in  a  glass-stoppered  bottle,  treated 
with  a  decinormal  solution  of  sodium  hydrate  until  the  reaction  is 
markedly  alkaline,  and  immersed  in  hot  water.  To  the  hot  fluid  a 
decinormal  solution  of  iodine  is  added  in  an  amount  Avhich  should 
exceed  that  of  the  alkali  solution  by  15  to  25  c.c.  The  l)ottlc  is 
now  closed  at  once,  shaken,  and  set  aside  until  cool.  The  solution 
is  then  acidified  Avith  dilute  sulphuric  acid,  and  the  excess  of  iodine, 
which  was  not  used  in  the  formation  of  tri-iodophenols,  retitrated  with 
a  decinormal  solution  of  sodium  thiosulphate.  One  c.c.  of  the  iodine 
solution  represents  1.567  milligramme  of  phenol,  or  1.8018  milli- 
gramme of  cresol.  As  the  latter  predominates  in  the  urine,  it  is  best 
to  express  the  results  in  terms  of  cresol.  Thus  modified,  the  method 
is  also  applicable  in  the  presence  of  sugar.  The  older  gravimetric 
method,  by  which  the  phenols  were  isolated  as  bromine  substitution- 
products,  has  now  been  largely  abandoned,  as  it  has  been  shown 
that  the  resulting  precipitate  is  not  of  constant  composition,  and 
contains  varial)le  amounts  of  dibromocresol,  besides  tribromophenol 
and  bromo-tril)romophenol. 

Indoxyl  Sulphate. — The  indoxyl  sulphate  which  occurs  in  the 
urine  in  combination  with  potassium  and  sodium  is  usually  spoken 
of  as  indlcan,  but  sh(^uld  not  be  confounded  with  the  vegetal)le  indi- 
can,  which,  as  has  been  shown,  is  a  glucoside  of  the  composition 
CofiHojNOiy.  The  amount  which  is  daily  excreted  by  man  is  nor- 
mally small,  and  corresponds  to  about  0.0066  gramme.  I-iarger 
quantities  are  observed  when  from  any  cause  intestinal  putrefaction 


270  THE   URINE. 

is  increased,  or  in  cases  in  which  putrefactive  changes  are  taking 
place  in  the  body  proper,  as  in  empyema,  providing  that  active 
resorption  can  occur. 

In  herbivorous  animals  larger  amounts  are  found  than  in  the 
carnivora.  Artificially  an  increased  elimination  can  be  effected  by 
feeding  animals  with  ortho-nitro-phenyl-propiolic  acid,  Avhich  is 
reduced  in  the  body  to  indoxyl,  according  to  the  equation  : 

/C=COOH  /COH^CH 

CfiH  /  +  8H  =  QH/  +  3H,0 

Ortho-nitro-phenyl-  Indoxyl. 

propiolic  acid. 

Indican  crystallizes  in  colorless  platelets,  which  are  readily  soluble 
in  water  and  hot  alcohol,  while  in  cold  alcohol  they  dissolve  with 
great  difficulty.  On  decomposition  with  hydrochloric  acid  the 
indoxyl  is  obtained  in  the  form  of  oily  droplets  of  an  exceedingly 
offensive,  feculent  odor.  On  oxidation  this  is  then  transformed  into 
indigo-blue,  as  is  shown  in  the  equation  : 

/C.OH  --=  CH  /CO  .  /CO  V 

2C6H/  +20  =  C6H,<         >C^C<         XH, +  2H,0 

Indoxyl.  Indigo-blue. 

On  heating  an  aqueous  solution  of  indoxyl  to  a  temperature  of 
130°  C.  indoxyl-red  also  results.  This  is  a  brown  amorphous  sub- 
stance, which  is  insoluble  in  water,  but  dissolves  with  ease  in 
alcohol,  ether,  and  chloroform,  with  a  beautiful  red  color.  When 
Jaffe's  test  for  indican  is  applied  to  the  urine,  or  when  this  is  boiled 
and  treated  drop  by  drop  with  concentrated  nitric  acid  (Rosenbach's 
reaction ),  a  mixture  of  a  blue  and  a  red  pigment  is  not  infrequently 
obtained,  and  it  is  quite  likely  that  the  latter  is  in  part  at  least  refer- 
able to  the  formation  of  the  indoxyl-red.  According  to  some  observ- 
ers, the  chromogen  of  this  substance  is  identical  with  the  so-called 
urohsematin,  and  the  pigment  is  ])robably  the  same  as  the  red  pig- 
ment of  Scherer,  the  urrhodin  of  Heller,  the  urorubin  of  Plosz,  the 
indirubin  of  Schunk,  the  indigo-purpurin  of  Bayer,  the  pigment 
of  Giaco,sa  and  others. 

The  blue  pigment  which  is  found  together  with  the  red  pigment 
when  urine  is  treated  with  a  strong  mineral  acid  and  an  oxidizing 
agent  is,  as  has  been  indicated,  indigo-blue,  and  is  identical  with 
urocyanin,  cyanurin,  Harnblau,  uroglaucin,  etc.,  of  former  observers. 
As  a  general  rule,  its  amount  is  far  greater  than  that  of  the  red  pig- 
ment, and  is  at  times  the  only  one  that  is  obtained.  In  other  cases, 
however,  the  red  seems  to  prevail,  and  in  still  others  both  are  appar- 
ently present  in  about  equal  proportion.  The  cause  of  those  varia- 
tions is  as  yet  not  understood,  but  probably  rests  upon  variations  in 
bacterial  action  in  the  intestinal  tract.  As  a  general  rule,  indeed, 
notable  quantities  of  the  red  pigment  are  observed  only  under  patho- 
logical conditions. 


TEE  AROMATIC  CONSTITUENTS  OF  THE   URINE.         271 

Tests  for  Indican. — All  the  tests  eraployed  for  the  purpose  of 
demonstrating  the  presence  of  indican  in  the  urine  are  essentially 
based  upon  the  decomposition  of  the  substance,  with  the  liberation 
of  indoxyl  and  its  oxidation  to  indigo-blue. 

Jaffe's  Test,  as  modified  by  Stokvis. — A  few  cubic  cen- 
timeters of  urine  are  treated  with  an  equal  volume  of  concentrated 
hydrochloric  acid,  and  two  or  three  drops  of  a  strong  solution  of 
sodium  hypochlorite.  The  indigo-blue  which  thus  results  is  then 
extracted  by  shaking  with  a  little  chloroform.  If  red  pigment  has 
been  formed  at  the  same  time,  the  color  varies  from  a  violet  to  a 
purplish  red. 

If  it  is  desired  to  separate  the  two  pigments,  the  chloroform  extract 
is  evaporated  to  dryness  and  the  residue  washed  with  a  mixture  of 
equal  ])arts  of  96  per  cent,  alcohol,  ether,  and  Mater.  This  dissolves 
the  red  pigment  and  leaves  the  indigo-blue  behind.  Care  must  be 
had,  however,  not  to  add  too  much  of  the  hypochlorite  solution,  as 
otherwise  the  indigo-blue  is  oxidized  to  isatin,  and  no  color  at  all  is 
obtained.  Should  this  happen  after  the  addition  of  only  one  or 
two  drops,  the  following  test  had  better  be  employed,  as  a  further 
oxidation  is  here  not  effected  : 

Obeema  yer's  Test. — A  few  cubic  centimeters  of  urine  are  treated 
with  an  equal  volume  of  a  2  pro  mille  solution  of  ferric  chloride  in 
concentrated  hydrochloric  acid.  The  indigo-blue  is  extracted,  as 
above,  by  shaking  with  a  little  chloroform.  As  in  the  above  test, 
indoxyl-red  may  thus  also  be  obtained,  and  is  separated  from  the 
blue  pigment  as  just  described. 

Test  for  Urohsematin  (so-called). — A  small  amount  of  urine  is  thor- 
oughly agitated  with  chloroform  and  allowed  to  stand  for  a  few 
days.  The  chromogen  of  indoxyl-red  is  thus  extracted,  for  on  adding 
a  drop  of  concentrated  hydrochloric  acid  to  the  chloroform  extract 
a  beautiful  rose-color  appears,  which  varies  in  intensity  with  the 
amount  of  tlie  chromogen  present.  In  normal  urine  a  faint  reaction 
only  is  usually  seen  ;  but  in  disease,  and  notably  in  ileus,  peritonitis, 
and  cancer  of  the  stomach,  I  have  repeatedly  met  with  more  indigo- 
red  than  indigo-blue. 

Rosenbach's  Reaction. — This  reaction  is  mostly  obtained  under 
pathological  conditions,  and  indicates  the  existence  of  greatly  in- 
creased intestinal  putrefaction.  It  is  referable  to  the  simultaneous 
formation  of  indigo-red  and  indigo-blue. 

While  boiling,  a  few  cubic  centimeters  of  urine  are  treated  drop 
by  drop  with  concentrated  nitric  acid  containing  a  little  nitrous 
acid,  when  in  the  presence  of  much  indigo-red,  viz.,  its  chromogen, 
the  urine  assumes  a  dark  Burgundy  color,  and  usually  shows  a 
bluish  tint  when  held  to  the  light.  On  standing,  the  red  pigment 
is  precipitated.  If  much  indigo-blue  is  present  at  the  same  time, 
as  is  usual,  the  foam  of  the  liquid  is  colored  blue.  On  adding  an 
excess  of  the  acid  the  color  often  disappears  and  the  urine  turns 
yellow. 


272  THE  URINE. 

The  isolation  of  indican  from  urine  as  such  is  a  rather  complicated 
process,  and  need  not  be  described  at  this  place. 

Quantitative  Estimation. — Wang's  Method  ( modified  by  Ellinger). 
— This  method  is  based  upon  the  decomposition  of  the  indican  by 
strong  hydrochloric  acid,  and  the  oxidation  of  the  resulting  indoxyl 
to  indigo-blue.  This  is  then  transformed"  into  indigo-sulphuric 
acid,  and  estimated  as  such  by  titration  with  a  solution  of  potassium 
permanganate  of  known  strength.  To  this  end,  a  stock  solution  of 
the  salt  is  kept  on  hand,  which  contains  about  3  grammes  to  the 
liter.  Five  c.c.  of  this  solution  are  diluted  with  195  c.c.  of  Avater, 
when  the  titre  is  ascertained  before  each  titration  by  comparing  it 
M'ith  a  dilute  solution  of  oxalic  acid.  The  amount  of  indigo-blue 
which  each  cubic  centimeter  represents  is  ascertained  by  multiplying 
the  corresponding  amount  of  oxalic  acid  by  1.04. 

The  amount  of  urine  which  is  necessary  varies  with  the  amount 
of  indican  present.  If  a  preliminary  test  gives  an  intense  reaction, 
from  25  to  50  c.c.  are  sufficient ;  otherwise  it  is  better  to  use  larger 
amounts,  as  from  200  to  250  c.c.  The  urine,  which  should  be  acid, 
is  then  ])recipitated  with  a  20  per  cent,  solution  of  basic  lead  acetate, 
care  being  taken  to  avoid  an  excess.  A  large  portion  of  the  filtrate, 
representing  a  known  amount  of  urine,  is  then  treated  with  an 
equal  volume  of  Obermayer's  reagent,  and  extracted  with  chloro- 
form by  shaking.  This  is  continued  with  portions  of  30  c.c.  until 
the  chloroform  takes  up  no  more  coloring-matter.  The  combined 
extracts  are  freed  from  chloroform  by  distillation.  The  residue  is 
dried  for  a  few  minutes  on  a  water-bath,  and  is  then  washed  with 
hot  water.  The  solution  is  passed  through  a  small  filter,  so  as  to 
collect  any  particles  of  the  blue  pigment  which  may  be  present  in 
suspension.  The  filter  is  dried,  extracted  Avith  boiling  chloroform, 
and  the  resulting  solution  filtered  into  the  flask  containing  the 
residual  indigo-blue.  The  chloroform  is  distilled  off,  and  the  resi- 
due treated  with  3  or  4  c.c.  of  concentrated  sulphuric  acid,  while  still 
warm.  This  solution  is  kept  on  the  water-bath  for  five  to  ten  min- 
utes. It  is  then  poured  into  100  c.c.  of  water,  the  bottle  is  washed 
out  with  a  little  more  water,  when  the  solution  and  w^ashings  are 
filtered  and  titrated  with  the  permanganate  solution.  The  color  at 
first  changes  to  green,  and  finally  the  solution  becomes  yellowish  or 
colorless.     The  calculation  is  conducted  as  outlined  above. 

Skatoxyl  Sulphate. — Skatoxyl  sulphate,  like  indoxyl  sulphate, 
occurs  in  the  urine  in  combination  with  potassium  and  sodium.  Its 
amount,  however,  is  normally  small,  and  it  may  at  times  be  absent 
altogether.  Larger  quantities  are  found  under  pathological  condi- 
tions associated  with  an  increased  degree  of  intestinal  putrefac- 
tion, and  it  may  then  happen  that  more  skatoxyl  suljihate  is  found 
than  indican.  This,  however,  is  uncommon,  and  in  disea.se  also 
more  indican  is  usually  present.  Like  indican,  it  is  decomposed 
on  treating  Mith  concentrated  hydrochloric  acid,  and  on  subsequent 
oxidation  the  liberated  skatoxyl  yields  pigments  which  are  for  the 


THE  AROMATIC  CONSTITUENTS  OF  THE   URINE.         273 

most  part  of  a  red  color.  Of  their  chemical  nature,  however, 
nothing  is  known.  One  of  these  may  jx)ssibly  be  identical  M'ith 
Rosin's  urorosein.  Rosin,  to  be  sure,  claims  that  the  chromogen  of 
urorosein  is  not  a  conjugate  sulphate  but  we  know  that  a  portion  of 
the  skatol  also  appears  in  combination  with  glucuronic  acid  in  the 
urine,  and  it  is  hence  possible  that  his  pigment  may  be  derived  from 
this  source.  Urines  containing  notable  quantities  of  skatoxyl 
become  darker  on  exposure  to  the  air,  and  may  gradually  turn  a 
reddish-violet  or  almost  a  black  color.  This  change,  as  in  the 
phenol  urines,  begins  at  the  surface  and  gradually  extends  down- 
ward. 

Tests. — To  demonstrate  the  presence  of  skatoxyl  in  urine,  this 
is  strongly  acidified  with  hydrochloric  acid  and  extracted  with 
amyl  alcohol,  which  takes  up  the  coloring-matter.  Chloroform 
and  ether  do  not  dissolve  this  in  acid  solution,  but  do  so  in  neutral 
or  alkaline  solution,  providing  that  the  pigment  has  been  freshly 
formed. 

Test  for  Urorosein  (so-called). — A  few  cubic  centimeters  of  urine 
are  treated  with  an  equal  amount  of  concentrated  hydrochloric  acid 
and  one  or  two  dro])s  of  a  strong  solution  of  calcium  hypochlorite. 
The  indigo-blue  is  extracted  with  chloroform,  and  it  will  now  be 
observed  that  the  supernatant  fluid  presents  a  red  color.  On  shaking 
with  amyl  alcohol  this  takes  up  the  red  pigment,  which  is  thus 
manifestly  not  identical  with  indigo-red.  Upon  the  addition  of 
sodium  hydrate  to  the  alcoholic  solution  the  color  disappears,  but 
reappears  upon  the  subsequent  addition  of  hydrochloric  acid.  On 
standing,  the  color  gradually  disappears.  Under  normal  conditions 
this  reaction  is  not  w^ell  marked,  but  becomes  quite  distinct  in  cases 
in  which  intestinal  putrefaction  is  much  increased. 

A  portion  of  the  skatol,  as  I  have  already  stated,  appears  in  the 
urine  as  skafol-carbonic  acid: 

>C.COOH. 


The  substance  is  usually  present  in  exceedingly  small  amounts, 
however,  and  has  not  been  isolated  in  substance.  To  demonstrate 
its  presence  even,  it  is  necessary  to  work  with  several  liters  of 
urine.  To  this  end,  the  fluid  (5000-6000  c.c.)  is  evaporated  to  a 
syrup ;  the  residue  is  extracted  with  absolute  alcohol,  the  alcoholic 
solution  is  evaporated,  and  the  remaining  material  extracted  with 
ether  after  acidifying  strongly  w'ith  sulphuric  acid.  From  the 
ethereal  solution  the  substance  is  obtained  in  aqueous  solution  by 
shaking  with  a  dilute  solution  of  sodium  hydrate.  The  alkaline 
solution  ii^  then  evaporated  and  the  residue  extracted  with  alcohol. 
This  extract  is  now  concentrated  to  about  100  c.c.  and  precipitated 
with  an  equal  volume  of  ether.  The  filtrate  is  evaporated  to  dry- 
ness, the  residue  acidified  with  hydrochloric  acid,  and  extracted  with 

IS 


274  THE   URINE. 

ether.  The  residue  of  the  ethereal  extract  is  then  finally  dissolved 
in  hot  water.  To  remove  the  remaininji:  hydrochloric  acid,  this 
solution,  after  filtering  and  cooling,  is  again  evaporated  to  dryness 
and  redissolved  in  hot  water.  To  tlcmunstrate  the  presence  of 
skatol-carbonic  acid,  a  portion  of  this  final  solution  is  then  treated 
with  a  few  drops  of  pure  nitric  acid  and  a  trace  of  potassium 
nitrite.  A  red  color  thus  develops,  which  may  be  extracted  with 
amyl  alcohol  or  acetic  ether.  This  solution  shows  a  band  of  absorp- 
tion in  the  green  portion  of  the  spectrum.  On  adding  a  solution  of 
sodium  hydrate  to  the  ethereal  solution  the  red  color  disappears,  btit 
reappears  on  the  subsequent  addition  of  hydrochloric  acid. 

In  addition  to  the  aromatic  bodies  which  have  thus  far  been  con- 
sidered, traces  of  the  aromatic  oxy-acids  may  also  appear  in  the 
urine  in  combination  with  sulphuric  acid,  but  the  amount  is  exceed- 
ingly small,  and  may  well  be  ignored. 

The  Conjugate  Glucuronates. 

Glucuronic  acid  as  such  does  not  occur  in  the  urine.  The  sub- 
stance can  combine  with  various  aromatic  bodies,  however,  and  may 
in  this  manner  escape  further  oxidation.  Normally  it  is  found  only 
in  traces  (approximately  0.004  per  cent.),  in  combination  with 
indoxyl,  skatoxyl,  and  especially  with  phenol,  while  the  greater  por- 
tion of  these  bodies  is  eliminated  in  the  form  of  conjugate  sulphates, 
as  already  described.  Larger  amounts  of  conjugate  glucuronates 
are  found  in  the  urine  after  the  administration  of  chloral,  cam})hor, 
naphthol,  terpene,  borneol,  menthol,  toluol,  euxanthin,  morphin, 
antipyrin,  and  numerous  alcohols  and  ketones.  The  resulting  com- 
pounds are  closely  related  to  the  glucosides.  So  far  as  the  hydrox- 
ylated  compounds  are  concerned,  these  are  apparently  capable  of 
uniting  with  glucuronic  acid  (or  sulphuric  acid)  to  form  conjugate 
glucuronates  (or  sulphates)  without  previous  oxidation,  while  this  is 
not  the  case  with  compounds  of  the  same  composition,  in  which, 
however,  the  oxygen  is  present  in  ketone  form.  At  the  same  time 
it  is  an  open  question  whether  the  union  of  such  alcohols  with  the 
acids  occurs  directly,  or,  as  is  possibly  the  case  with  glucuronic  acid, 
with  antecedents  of  the  same,  and  that  the  resulting  compounds  are 
subsequently  oxidized  to  glucuronates  during  their  passage  through 
the  organism. 

In  part  at  least  the  synthesis  of  the  conjugate  glucuronates  occurs 
in  the  liver,  and  in  some  cases  their  appearance  is  manifestly  the 
expression  of  a  poison-destruction  on  the  part  of  the  organism.  This 
explanation  does  not  hold  good  in  all  cases,  however.  Following 
the  ingestion  of  glucose  in  large  amounts  or  in  diabetes  the  appear- 
ance of  glucuronates  is  evidence  primarily  of  deficient  oxidation. 
We  may  here  imagine  that  the  oxidation  of  glucose  to- glucuronic 
acid  (see  below)  pursues  a  normal  course,  but  that  its  further  de- 
struction is  impeded.    The  larger  amount  of  the  circulating  glucuro- 


THE  AROMATIC  CONSTITUENTS   OF  THE    URINE.         275 

iiic  acid  then  combines  with  phenol,  indoxyl,  and  skatoxyl,  which 
would  otherwise  have  united  with  sulphuric  acid,  and  as  a  result  we 
find  a  diminished  excretion  of  conjugate  sulphates.  Any  glucuronic 
acid  remaining  is  probably  oxidized  to  oxalic  acid.  This  explains 
satisfactorily  the  oxaluria  which  is  frequently  observed  in  diabetes 
and  which  follows  the  ingestion  of  large  amounts  of  glucose.  Ac- 
cording to  the  character  of  the  aromatic  component  uniting  with 
glucuronic  acid,  the  resulting  compounds  have  been  termed  camphor- 
glucuronic  acid,  urochloralic  acid,  menthol-glucuronic  acid,  phenyl-, 
indoxyl-,  skatoxyl-glucuronic  acid,  etc. 

Of  the  origin  of  the  glucuronic  acid  little  is  known.  That  it  is 
formed  in  the  tissues  of  the  body  is  apparent  from  the  fact  that  even 
in  a  starving  animal  the  administration  of  camphor,  chloral,  etc., 
leads  to  elimination  of  these  substances  in  combination  with  the  acid 
in  question.  As  glucuronic  acid  is  a  derivative  of  glucose,  we  may 
imagine  that  during  starvation  it  is  derived  from  glycogen,  or  even 
from  the  albumins.  It  has  been  demonstrated,  as  a  matter  of  fact, 
that  the  formation  of  glycogen  in  the  liver  can  be  artificially  in- 
creased by  introducing  glucuronic  acid  with  the  food.  The  chemi- 
cal relation  of  glucuronic  acid  to  glucose  has  already  been  considered. 
On  oxidation  glucose  thus  first  yields  the  monobasic  gluconic  acid, 
and  then  the  dibasic  saccharinic  acid.  The  latter  in  turn  may  be 
transformed  into  saccharo-lactonic  acid,  which  on  reduction  yields 
glucuronic  acid,  so  that  this  stands  midway  between  gluconic  acid 
and  saccharinic  acid.     These  relations  are  shown  by  the  formulae  : 

CH.,.OH  —  (CH.0H)4  —  COH,  glucose. 

CH^.OH  —  (CH.OH)^  —  COOH,  gluconic  acid. 

COOH     —  (CH.0H)4  —  COH,  glucuronic  acid. 

COOH     —  (CH.OH)^  —  COOH,  saccharinic  acid. 

On  boiling  with  water  glucuronic  acid  is,  in  part  at  least,  trans- 
formed into  its  anhydride,  glucuron,  CgHgOg. 

Formerly  glucuronic  acid  was  also  thought  to  be  derived  from 
chondroitin-sulphuric  acid  ;  this  view,  however,  has  now  been  aban- 
doned. 

Glucuronic  acid  has  thus  far  not  been  obtained  in  crystalline 
form.  It  is  a  syrupy  substance  which  is  readily  soluble  in  water 
and  alcohol.  Its  anhydride,  however,  is  a  crystalline  body,  and  is 
likewise  soluble  in  water  but  insoluble  in  alcohol.  The  free  acid 
and  its  alkaline  salts  are  dextrorotatory,  while  the  conjugate  glucuro- 
nates  turn  the  polarized  light  to  the  left.  The  free  acid,  moreover, 
as  well  as  its  salts  and  most  of  its  compound  ethers,  reduces  the 
oxides  of  copper,  bismuth,  and  silver  in  alkaline  solution,  and  it  is 
thus  possible  to  confound  them  with  glucose  if  reliance  is  placed  upon 
the  corresponding  tests  alone.  With  phenyl-hydrazin  the  free  acid 
is  said  to  form  a  crystalline  compound  with  a  melting-point  of  114°- 
115°  C.  Unlike  glucose,  it  is  non-fermentable.  It  gives  the  fur- 
furol  reaction  and  simulates  the  pentoses  in  reacting  with  phloro- 


276  THE   URINE. 

glucin  hydrochlorate,  but  not  with  orcin  (see  page  306).  The  amount 
of  furt'urol  wliich  may  be  obtained  from  glucuronic  acid  on  distilla- 
tion with  hydrochloric  acid  is  quite  considerable.  According  to 
Giinther,  Chalniot,  and  Tollens,  it  yields  46  per  cent.,  and  its  deriva- 
tives, euxxanthinic  acid  and  urochloralic  acid,  12.5  and  17  per  cent, 
respectively. 

The  demonstration  of  the  presence  of  glucuronic  acid  in  the  urine 
and  other  fluids  of  the  body  directly  is  most  conveniently  conducted 
bv  decomposing  the  conjugate  glucuronates  with  1  per  cent,  sul- 
phuric acid  in  the  autoclave,  and  preparing  its  p-bromphenvl- 
hvdrazin  compound  (COH.(CH.OH),.COOH.NH2.NH.C,H,Br). 
This  is  characterized  by  its  high  melting-point,  236°C.  (200°-216^C.) 
in  impure  form)  its  insolubility  in  absolute  acohol,  and  its  high  degree 
of  hevorotation  in  a  pyridin-alcoholic  solution,  viz.,  7°25'.  The 
same  method  may  be  used  for  its  quantitative  estimation  ( Zeit.  f. 
phijs.  Chem.,  1900,  vol.  xxix.,  p.  256). 

For  the  clinical  recognition  of  glucuronic  acid  compounds  the  fol- 
lowing data  suffice :  kevorotation  of  the  urine  after  fermentation, 
which  diminishes  on  boiling  with  acids  or  changes  to  dextrorotation  ; 
an  increased  reduction  after  boiling  with  dilute  sulphuric  acid,  and 
a  positive  orcin  reaction  (see  Pentoses),  which  was  negative  before 
boiling. 

The  Compound  Glycocolls. 

As  has  been  pointed  out,  phenyl-propionic  acid  and  phenyl-acetic 
acid,  which  are  both  formed  from  albuminous  material  during  the 
process  of  intestinal  putrefaction,  are  in  part  absorbed  in  the  intestinal 
tract,  and  are  eliminated  in  the  urine  in  combination  with  glycocoU 
as  hippuric  acid  and  phenaceturic  acid,  respectively.  But  while 
phenyl-acetic  acid  unites  with  glycocoll  directly,  phenyl-propionic 
acid  is  usually  first  oxidized  to  benzoic  acid  (see  page  95). 

Hippuric  Acid. — While  a  certain  amount  of  the  benzoic  acid 
wliich  enters  into  the  construction  of  the  hippuric  acid  molecule  is 
derived  from  the  plienyl-propionic  acid  which  results  during  the 
process  of  intestinal  putrefaction,  another  portion  is  ingested  as 
such,  or  in  the  form  of  other  aromatic  substances  which  can  be 
transformed  into  benzoic  acid  in  the  animal  body.  We  can  thus 
understand  why  larger  amounts  of  hippuric  acid  are  encountered 
in  the  urine  of  herbivorous  animals  than  in  that  of  the  carnivora,  as 
the  food  of  the  former  always  contains  very  considerable  amounts  of 
toluol,  cinnamic  acid,  quinic  acid,  etc.,  all  of  which  may  give  rise  to 
benzoic  acid.  In  man  the  daily  elimination  corresponds  to  about 
0.7  gramme,  but  may  be  increased  by  the  ingestion  of  such  articles 
of  food  as  cranberries,  prunes,  reine-claudes,  etc. 

In  a  few  instances  the  substance  has  been  found  ,in  urinary 
sediments. 

While  the  source  of  the  aromatic  component  of  hippuric  acid 
is  thus  quite  well  understood,  we  know  but  little  of  the  origin  of 


THE  AROMATIC  CONSTITUENTS  OF  THE   URINE.         Til 

glycocoll.  To  a  certain  extent  tliis  may  also  be  formed  during  the 
process  of  albuminous  putrefaction,  as  we  know  that  all  albumins 
contain  a  glycocoll  radicle,  but  there  is  reason  to  believe  that  it 
may  likewise  originate  within  the  tissues  of  the  body.  It  mani- 
festly plays  an  important  role  in  the  process  of  metabolism,  for  we 
have  seen  that  to  a  certain  extent,  no  doubt,  it  is  concerned  in  the 
formation  of  urea,  and  it  has  also  been  noted  that  in  man  and  in 
other  animals  a  very  considerable  proportion  of  cholalic  acid  is  elimi- 
nated from  the  body  in  combination  with  glycocoll. 

Through  the  interesting  researches  of  Schmiedeberg  we  know  that 
the  synthesis  of  glycocoll  and  benzoic  acid  is  in  dogs  effected  in  the 
kidneys  exclusively.  In  other  animals,  however,  this  process  may 
occur  in  other  organs  as  well,  for  it  has  been  shown  that  after 
removal  of  the  kidneys  hippuric  acid  can  be  isolated  from  the  liver 
and  the  muscles,  at  least,  if  benzoic  acid  has  been  previously 
administered. 

Properties. — Hippuric  acid  crystallizes  in  long  rhombic  prisms 
when  allowed  to  separate  from  its  solutions  slowly,  while  when 
rapidly  formed,  and  especially  if  the  amount  is  small,  it  occurs  in 
long  needles  which  are  frequently  grouped  in  stnr«  ■^.nd  rosettes. 
The  melting-point  of  the  substance  is  187.5°  C.  It  is  soluble  with 
great  difficulty  in  cold  water  and  ether,  while  in  hot  water  and 
alcohol  it  dissolves  with  comparative  ease.  In  aqueous  solutions  of 
the  alkaline  hydrates  and  carbonates  it  dissolves  with  the  formation 
of  the  corresponding  salts,  from  which  the  free  acid  may  again  be 
obtained  by  acidifying  with  a  mineral  acid. 

On  boiling  with  dilute  mineral  acids  or  alkalies  hippuric  acid  is 
decomposed  into  its  components.  The  same  result  is  reached  if 
ammoniacal  decomposition  is  allowed  to  occur,  and  in  such  urines 
benzoic  acid  only  is  found.  In  traces,  benzoic  acid  is  said  to  occur 
in  every  urine  together  with  hippuric  acid,  and  it  is  thought  that  its 
presence  under  normal  conditions  may  be  due  to  the  action  of  a 
ferment,  the  so-called  Iddcnzyme  of  Schmiedeberg,  which  has  been 
found  in  the  kidneys,  and  which  is  known  to  be  capable  of  effecting 
the  decomposition  of  hi})puric  acid  as  outlined. 

On  heating  hippuric  acid  in  a  dry  test-tube  it  melts,  and  is  then 
decomposed  with  the  formation  of  benzoic  acid,  which  sublimes  in 
the  upper  portion  of  the  tube.  The  liquid  mass  at  the  same  time 
assumes  a  red  color,  and  develops  an  odor  wiiich  at  first  is  suggestive 
of  hay,  but  subsequently  resembles  that  of  hydrocyanic  acid.  This 
reaction,  together  v/ith  the  form  of  the  crystals  and  tlieir  insolubility 
in  petroleum-ether,  serves  to  distinguish  hippuric  acid  from  benzoic 
acid.  But  like  this,  it  develops  a  marked  odor  of  bitter  almonds 
when  it  is  evaporated  with  nitric  acid  and  the  residue  is  then 
heated.  The  reaction  is  due  to  the  formation  of  nitrobenzol. 
In  the  urine  hippuric  acid  is,  of  course,  not  present  in  the  free 
state,  but  in  combination  with  alkalies,  and  notably  potassium  and 
sodium. 


278  THE   URINE. 

Synthesis  of  Hippuric  Acid. — Hippuric  acid  can  be  formed  syn- 
tlieticallv  in  vitro  also  from  benzoic  acid  and  glycocoll  by  healing 
the  two  substances  together  at  a  temperature  of  160°  C.  in  a  sealed 
tube.  In  a  similar  manner  it  is  obtained  from  benzamide  and 
monochlor-acetic  acid.  The  reactions  which  take  place  may  be 
represented  by  the  equations  : 

CeH^.COOH     +  CH.,(NH2).C00H  =  CH2.NH(C6H,,CO).COOH  +  H^O 
Benzoic  Glycocoll.  Hippuric  acid, 

acid. 

C6H5.CO.NH2  +  CH.,.  Cl.COOH        =  CH^.  NH(  QHs.CO ).  CX)OH  +  HCl 

Benzamide.  Jibnochlor-  Hippuric  acid, 

acetic 
acid. 

Isolation  of  Hippuric  Acid. — Hippuric  acid  is  most  conveniently 
isolated  from  the  urine  of  herbivorous  animals,  in  which,  as  has 
.  been  stated,  it  is  present  in  much  greater  amounts  than  in  that  of 
man  and  the  carnivorous  animals.  To  this  end,  1000  c.c.  of /rcsA 
urine  are  rendered  feebly  alkaline  w'th  milk  of  lime  and  boiled  for  a 
few  minutes.  The  liquid  is  filtered  while  still  warm,  concentrated, 
and  on  cooling  acidified  with  hydrochloric  acid  added  in  moderate 
excess.  After  twentv-four  hours  the  crystals  of  hippuric  acid  which 
have  separated  out  are  filtered  off,  and  are  freed  from  adhering  pig- 
ments as  follows  :  they  are  dissolved  in  hot  water,  and  treated  with 
alum  and  then  with  an  amount  of  sodium  carbonate  sufficient  to 
cause  the  formation  of  an  abundant  precipitate;  the  reaction,  how- 
ever, should  remain  still  acid.  The  pigments  are  retained  by  the 
aluminous  precipitate.  The  filtrate  is  then  strongly  concentrated, 
acidified  with  hydrochloric  acid,  and  allowed  to  stand.  In  this 
manner  the  hippuric  acid  is  obtained  in  fairly  pure  form,  and  can  be 
further  purified,  if  desired,  by  reerystallization  from  hot  water. 

Isolation  of  Glycocoll  and  Benzoic  Acid  from  Hippuric  Acid. — As 
stated  before,  glycocoll  is  most  conveniently  obtained  from  hippuric 
acid.  To  effect  the  decomposition,  this  is  boiled  for  ten  to  twelve 
hours  with  four  parts  of  diluted  sulphuric  acid.  On  cooling,  the 
benzoic  acid  which  has  separated  out  is  filtered  off.  The  filtrate  is 
concentrated  and  extracted  with  ether,  which  takes  up  the  remain- 
ing benzoic  acid.  The  sulphuric  acid  is  now  removed  by  means  of 
barium  hydrate  and  the  excess  of  barium  precipitated  with  carbon 
dioxide.  '  The  filtrate  is  concentrated,  when  on  cooling  the  glycocoll 
is  obtained  in  crystalline  form. 

Isolation  and  Quantitative  Estimation. — Five  hundred  to  one  thou- 
sand c.c.  of  urine  are  rendered  slightly  alkaline  with  .sodium  car- 
bonate, and  are  evaporated  to  a  thick  syrup,  taking  care  that  the 
reaction  remains  alkaline.  It  is  then  extracted  with  cold  strong 
alcohol  (90  to  95  per  cent.)  by  adding  about  one-half  the  voltune  of 
the  original  solution,  and  allowing  the  mixture  to  stand  for  twenty- 
four  hours.  It  is  then  filtered  and  the  alcohol  distilled  off.  The 
remaining  aqueous  solution  is  acidified  with  dilute  sulphuric  acid, 
and  the  liberated  hippuric  acid  extracted   with  several  portions  of 


THE  AROMATIC  OXY-ACIDS.  279 

acetic  ether.  The  ethereal  solution  is  evaporated  to  dryness,  when 
the  remaining  impurities,  such  as  phenols,  aromatic  oxy-acids, 
benzoic  acid,  and  fat,  are  removed  by  washing  with  cold  petroleum- 
ether,  in  which  hippuric  acid  is  insoluble.  It  is  then  dissolved  in 
warm  water,  and  the  solution  evaporated  at  a  temperature  of  from 
50°  to  60°  C  until  crystallization  occurs.  On  cooling,  the  crystals 
are  filtered  oif  and  weighed.  The  mother-liquor  is  extracted  with 
acetic  ether,  the  ethereal  solution  is  evaporated  to  dryness,  and  the 
weight  of  the  residue  added  to  that  of  the  crvstals. 

Phenaceturic  Acid. — In  small  amounts  phenaceturic  acid  has 
been  repeatedly  obtained  from  the  urine  of  man,  but  is  principally 
met  with  in  that  of  herbivorous  animals,  in  which  the  putrefactive 
processes,  owing  to  the  greater  length  of  the  intestinal  tract  and  the 
character  of  the  food,  are  more  extensive.  On  boiling  with  dilute 
mineral  acids  it  is  decomposed  into  its  components,  as  shown  in  the 
equation  : 

CH^.NHICH,,  CsH-.COVCOOH  +  Hp  =  rH,,(C6H,).C00H  +  CHofNH^^.COOH 
Phenaceturic  acid.  Phenyl-acetic  acid.  "  GlycocoU. 

Properties. — Phenaceturic  acid  crystallizes  in  small  rhoml)ic  plates 
with  rounded  angles,  which  are  very  similar  to  the  corresponding 
crystals  of  uric  acid. 

Isolation. — Phenaceturic  acid  may  be  i.^^olated  from  the  urine  of 
the  horse  after  sej'taration  of  the  hippuric  acid,  as  shown  above. 
The  mother-liquor  is  then  extracted  with  acetic  ether,  which  takes 
up  the  acid.  On  evaporation  the  residue  is  dissolved  in  a  dilute 
solution  of  sodium  hydrate.  From  this  solution  the  substance  is 
again  extracted  with  ether,  after  acidifying  with  hydrochloric  acid, 
and  thus  purified,  and  finally  allowed  to  crystallize  from  its  aqueous 
solution. 

In  this  connection  brief  reference  may  be  made  to  omithuric  acid, 
which  may  be  obtained  from  the  urine  of  birds  when  these  are  fed 
with  benzoic  acid,  and  which  probably  also  represents  a  normal  con- 
stituent of  such  urine.  Its  formation  is  analogous  to  that  of  hip- 
puric acid  in  mammals,  but  the  benzoic  acid  here  combines  with  a 
diamido-acid,  ornithin,  which  is  a,  o-diamido-valerianic  acid,  as  has 
been  shown  before. 

The  relation  of  ornithin  to  arginin  has  already  been  considered. 

"THE   AROMATIC    OXY-ACIDS. 

The  aromatic  oxy-acids  which  may  be  found  in  the  urine  under 
normal  conditions  are  para-oxypheuyl-propionic  acid  or  hydro- 
paracumaric  acid,  and  para-oxyphenyl-acetic  acid,  which  results 
from  the  former  on  oxidation.  Both  are  derivatives  of  tyrosin, 
and  are  formed  during  the  process  of  intestinal  putrefaction,  as 
has  been  shown.  Para-oxyphenyl-lactic  acid  may  further  be 
encountered   when   tyrosin    has    been    administered    to  animals    in 


280 


THE   URINE. 


large  amounts.  Under  pathological  conditions,  as  in  acute  yellow 
atrophy,  where  leucin  and  tyrosin  may  a]>pear  in  the  urine  as  such, 
para-oxyphenyl-glycolic  acid  or  oxy-amygdalic  acid  has  also  been 
found.  Of  special  interest  finally  is  the  fact  that  in  some  instances 
dioxyphenyl-acetic  acid  (homogentisinic  acid)  and  trioxyphenyl-pro- 
pionic  acid  or  uroleucinic  acid  also  may  occur  in  the  urine.  The 
chemical  relationship  which  exists  between  these  various  substances 
and  tyrosin,  from  which  they  are  all  probably  derived,  is  apparent 
from  their  formulae : 


C«H. 


C6H,< 


/0H(1) 

"^CH2.CH(NH2).COOH(4)   para-oxyphenyl-amido-propionic  acid  (tyrosin). 

/0H(1) 

'~CH2.CH2.COOH(4)  para-oxyplienyl-propionic  acid. 


/ 


(0H)3 
CH,.CH,.COOH 


trioxyphenyl-propionic  acid. 


CeH,< 


/OH 

^CH2.CH(0H).C00H  para-oxyphenyl-lactic  acid. 


OH 
CH,.COOH 


para-oxyphenyl-acetic  acid. 


CfiH. 


C6H,< 


(OH), 


CHj.COOH  dioxyphenyl-acetic  acid. 

.OH 
-CH(OH).COOH        para-oxyphenyl-glycolic  acid. 


As  has  been  pointed  out,  the  two  aromatic  oxy-acids  which  are 
usually  found  in  the  urine  originate  in  the  intestinal  tract  during 
the  process  of  albuminous  putrefaction.  There  is  reason  to  believe, 
moreover,  that  homogentisinic  acid  and  uroleucinic  acid  are  likewise 
of  intestinal  origin,  and  are  referable  to  the  activity  of  a  special 
micro-organism,  which  is  usually  not  found.  But  as  para-oxy- 
phenyl-glycolic acid  manifestly  originates  in  the  tissues  of  the  body, 
we  must  admit  that  the  other  members  of  the  gri)up  may  also  be 
formed  beyond  the  intestinal  tract.  To  what  extent  this  occurs, 
however,  we  do  not  know. 

A  certain  fraction  of  these  bodies  appears  in  the  urine  in  com- 
bination with  sulphuric  acid,  but  the  greater  portion  is  eliminated 
as  such,  viz.,  as  potassium  and  sodium  salts.  The  amount  of  the 
common  oxy-acids,  however,  is  always  small,  and  rarely  exceeds  0.03 
gramme  in  the  twenty-four  hours. 

To  demonstrate  the   presence  of   the  common   oxy-acids  in  the 


THE  AROMATIC  OXY- ACIDS.  281 

urine,  we  proceed  as  follows  :  500  c.c.  of  urine  are  strongly  acidified 
with  hydrochloric  acid  and  distilled  until  the  phenols,  viz.,  phenol 
and  paracresol,  have  passed  over.  This  can  be  recognized  by  test- 
ing the  distillate  from  time  to  time  with  Millon's  reagent.  On 
cooling,  the  remaining  fluid  is  thoroughly  extracted  with  ether, 
which  takes  up  the  oxy-acids  as  well  as  pyrocatechin  and  hydro- 
quinon.  To  separate  the  acids  from  the  latter,  the  ethereal  extract 
is  shaken  with  a  dilute  solution  of  sodium  carbonate.  The  acids  are 
thus  transformed  into  the  corresponding  salts  and  are  found  in  the 
alkaline  solution.  After  separation  from  the  ether  this  is  acidified 
with  dilute  sulphuric  acid  and  extracted  with  ether.  The  ethereal 
solution  contains  the  free  oxy-acids.  Their  presence  can  be  demon- 
strated by  evaporating  to  dryness,  when  the  residue  is  dissolved  in  a 
little  water  and  tested  with  Millon's  reagent. 

To  isolate  the  individual  acids,  much  larger  quantities  of  urine 
are  necessary.  We  may  then  proceed  as  described  in  the  section  on 
the  Feces. 

Homog"entisinic  Acid. — The  presence  of  homogentisinic  acid 
may  be  suspected  if  a  urine,  on  being  rendered  alkaline,  turns  a 
dark  reddish-brown  on  standing,  and  ultimately  becomes  black.  At 
the  same  time  a  positive  reaction  with  Fehling's  solution  is  obtained, 
while  polari metric  examination  shows  that  the  urine  is  optically 
inactive.  Nylander's  solution  is  not  reduced.  Upon  the  addition 
of  a  small  amount  of  a  dilute  solution  of  ferric  chloride  a  greenish- 
blue  color  develops,  which  is  only  of  momentary  occurrence,  how- 
ever. 

Boedeker  was  the  first  to  describe  a  urine  of  this  kind,  and  termed 
the  substance  giving  rise  to  the  above  reaction  alkapton.  Subse- 
quently, however,  he  expressed  the  opinion  that  his  alkapton  may 
have  been  pyrocatechin.  Other  investigators  have  isolated  sub- 
stances from  such  urines,  which  have  been  variously  termed  pyro- 
catechuic  acid,  urrhodinic  acid,  glycosuria  acid,  uroxanthinic  acid, 
and  uroleucinic  acid,  but  there  is  reason  to  suppose  that,  with  the 
possible  exception  of  the  last  mentioned,  all  these  substances  are 
identical  with  homogentisinic  acid.  This  was  first  isolated  from  an 
"  alkapton  "  urine  by  Baumann  and  Wolkow,  and  has  since  been 
found  in  every  case  that  has  been  examined  in  this  direction. 

Alkaptonuria,  though  it  may  occur  in  disease,  has  been  regarded  as 
the  expression  of  an  unusual  form  of  intestinal  putrefaction  which 
in  no  way  affects  the  health  of  the  individual.  Some  observers,  on 
the  other  hand,  look  upon  it  as  a  metabolic  abnormality,  and  it  must 
be  confessed  that  micro-organisms  have  thus  far  not  been  isolated 
from  the  intestinal  contents  of  such  cases  which  are  capable  of 
effecting  the  transformation  of  tyrosin  to  homogentisinic  acid  in 
vitro.  That  tyrosin  can  be  its  source  is  undoubted,  for  it  has  been 
shown  that  following  the  administration  of  this  substance  homo- 
gentisinic acid  appears  in  the  urine  in  greatly  increased  amount. 
Baumann  thus  noted  that  Mdiile  the  average  elimination  in  one  of 


282  THE   URINE. 

his  cases  amounted  to  4.6  grammes,  14  grammes  were  once  extracted 
in  the  twenty-four  hours  after  tyrosin  had  been  ingested.  It  is  to 
be  noted,  however,  that  tyrosin  is  a  member  of  the  para-series,  while 
homogentisinic  acid  belongs  to  the  ortho-series.  A  direct  transfor- 
mation of  the  one  into  the  other  can  accordingly  not  occur.  But 
we  may  imagine  that  the  hydroxyl  group  of  the  tyrosin  is  first 
removed  by  reduction,  and  that  the  benzol  radicle  is  then  oxidized 
again  in  two  para-positions. 

Phenyl-alanin,  like  tyrosin,  can  also  increase  the  elimination  of 
homogentisinic  acid  (in  one  case  89.32  per  cent,  of  the  ingested 
phenyl-alanin  reajipeared  as  homogentisinic  acid). 

Isolation. — Homogentisinic  acid  may  be  conveniently  isolated 
from  the  urine  according  to  the  method  suggested  by  Garrod.  The 
collected  nrine  of  twenty-four  hours  is  heated  nearly  to  the  boiling- 
point,  and  then  treated  with  5  or  6  grammes  of  neutral  lead  acetate 
in  substance  for  every  100  c.c.  of  the  urine.  As  soon  as  the  salt  is 
dissolved,  the  resulting  precipitate  is  filtered  oif  and  the  filtrate  set 
aside  in  the  cold  for  twenty-four  hours.  The  crystals  of  lead  homo- 
gentisinate  are  then  collected  on  a  filter  and  dissolved  in  hot  water. 
This  solution  gives  the  various  reactions  described  above.  To  isolate 
the  free  acid,  the  lead  compound  is  decomposed  with  hydrogen  sul- 
phide and  the  filtrate  carefully  evaporated  on  a  water-bath  until  the 
fluid  begins  to  darken,  when  it  is  further  concentrated  in  a  vacuum 
to  the  point  of  crystallization.  The  resulting  crystals  are  soluble  in 
water,  alcohol,  and  ether,  but  are  insoluble  in  chloroform,  benzol, 
and  toluol.     They  melt  at  146.5°-147°  C. 

In  rare  cases  uroleucmic  acid  also  can  appear  in  the  urine.  In 
its  general  reactions  it  resembles  homogentisinic  acid,  but  does  not 
give  the  iron  reaction  described  above.  Unlike  the  latter,  it  reduces 
Nylander's  solution  when  present  to  the  extent  of  0.5  per  cent,  or 
more, 

Inosit. — The  origin  and  chemical  constitution  of  inosit  will  be 
considered  elsewhere.  According  to  Hoppe-Seyler,  it  mav  occur  in 
the  urine  under  normal  conditions,  but  more  commonly  it  is  found 
in  diseases  which  are  associated  with  a  high  grade  of  polyuria,  such 
as  diabetes  insipidus,  diabetes  mellitus,  in  chronic  interstitial  nepli- 
ritis,  etc. 

To  demonstrate  its  presence  in  the  urine  large  amounts  are  con- 
centrated to  a  syrup,  which  is  then  extracted  with  four  times  its 
volume  of  alcohol  by  boiling.  On  cooling,  this  extract  is  treated 
with  an  excess  of  ether,  when  the  inosit  gradually  crystallizes  out 
and  may  be  recognized  by  its  special  tests  (see  page  387). 

The  remaining  aromatic  substances  which  have  been  found  in  the 
urine  are  the  kynurcnic  acid  and  urocaninic  acid  of  the  urine  of 
dogs,  and  the  so-called  lithuric  acid,  which  has  been  obtained  from 
the  urine  of  the  ox.     The  two  latter  have  thus  far  been  found   in 


THE  AROMATIC  OXY- ACIDS.  283 

only  one  instance  and  need  not  be  considered  at  this  place.     Their 
formulae  are  given  as  : 

C12H1.2N4O4,  urocaninic  acid. 

CjjHigNOg,  lithuric  acid. 

The  so-called  damalic  acid  and  damaluric  acid,  which  are  obtained 
from  the  urine  of  the  horse  and  tlie  cow,  probably  represent  a  mixt- 
ure of  benzoic  acid  and  volatile  fatty  acids. 

Kynurenic  Acid. — Kynurenic  acid  is  said  to  be  a  constant  con- 
stituent of  the  urine  of  dogs.  Its  mother-substance  is  manifestly 
an  albuminous  derivative,  as  the  amount  which  appears  in  the  urine 
is  largely  dependent  upon  the  quantity  of  albuminous  food  ingested. 
Its  elimination,  moreover,  continues  during  starvation,  although  the 
amount  is  then  reduced  to  a  minimum.  Its  formation  is  aj)pareutly 
not  influenced  by  the  degree  of  intestinal  putrefaction,  as  the  same 
amounts  are  excreted  when  ])utrefactive  processes  have  been  reduced 
to  the  lowest  level  by  the  administration  of  calomel  or  iodoform. 

Gliissner  and  Langstein  have  recently  shown  that  the  kynurenic 
acid  originates  from  a  substance  which  in  turn  results  from  the 
albumins  during  the  process  of  pancreatic  digestion,  but  of  the 
nature  of  this  antecedent  nothing  is  as  yet  known.  Whether  the 
substance  or  its  antecedent  is  also  formed  in  the  intermediary 
metabolism  remains  to  be  seen. 

Kynurenic  acid  is  now  regarded  as  j'-oxy-/9-quinoliu-carbonic  acid, 
and  is  decomposed  by  heat,  with  the  formation  of  carbon  dioxide 
and  a  basic  substance,  fcynurnn,  viz.  ^-oxyquiuolin.  On  reduction 
the  latter  is  transformed  into  quinolin.  The  changes  are  repre- 
sented by  the  equations  : 

(1)  C9H5N(OH).COOH     =     CgHfiNCOH)      +     CO., 

Kynurenic  acid.  Kynurin. 

(2)  CgHfiNlOH)  +    2H    =      CgH.N      +     H^O 

Kynurin.  Quinolin. 

On  oxidation  both  kynurenic  acid  and  kynurin  yielded  kynuric  or 
oxalyl-anthranilic  acid  : 

^NH.CO.COOH 

The   synthesis  of   kynurenic   acid    has   been    accomplished   by   R. 
Camps. 

Isolation. — To  isolate  kynurenic  acid  from  the  urine,  500  c.c.  are 
treated  with  hydrochloric  acid  in  the  proportion  of  4  c.c.  for  every 
100  c.c.  of  the  urine.  On  standing  for  forty-eight  hours  the  sub- 
stance in  question  crystallizes  out  together  with  uric  acid.  To  sepa- 
rate it  from  the  latter,  dilute  ammonia  is  added  to  the  crystalline 
precipitate.  The  uric  acid  remains,  and  from  its  ammoniacal  solu- 
tion the  kynurenic  acid  is  then  precipitated  by  acidifying  with  hydro- 
chloric acid. 


284  THE   URINE. 

The  crystals  are  soluble  in  alcohol  and  melt  at  253°  C.  On 
evaporating  a  bit  of  the  material  with  hydrochloric  acid  and  potas- 
sium chlorate  on  a  porcelain  plate  a  reddish  residue  is  obtained, 
which  principally  consists  of  tetrachloro-oxykynurin.  When  this 
is  moistened  with  ammonia  a  brownish-green  color  develops,  and  on 
standing  this  soon  passes  into  a  fine  emerald  green. 

The   Fatty   Acids. 

The  Volatile  Fatty  Acids.— The  volatile  fatty  acids  which  may 
be  isolated  from  any  urine,  and  which  are  especially  abundant  in 
that  of  herbivorous  animals,  are  normally  derived  from  the  intestinal 
tract,  where  they  are  principally  formed  during  the  process  of  carbo- 
hydrate fermentation  ;  a  certain  fraction,  however,  is  also  referable 
to  albuminous  putrefaction.  These  acids  are  acetic  acid,  formic  acid, 
propionic  acid,  and  butyric  acid. 

The  non-volatile  acids,  capric  acid  and  caprylic  acid,  have  further 
been  found  in  the  urine  of  herbivorous  animals. 

In  man  about  0.05  gramme  of  volatile  fatty  acids  is  excreted  in 
twenty-four  hours.  Especially  large  amounts,  such  as  3  grammes  pro 
die,  have  been  found  in  the  urine  of  the  goat.  In  various  diseases 
larger  amounts  have  also  been  encountered  in  man,  but  it  is  still  an 
open  question  whether  they  are  then  derived  from  the  intestinal 
tract  exclusively.  From  decomposing  urine  a  larger  quantity  can 
be  obtained  than  from  fresh  urine.  This  is  no  doubt  owing  to  the 
fact  that  every  urine  contains  a  small  amount  of  carbohydrates, 
which  yield  fatty  acids  on  bacterial  decomposition.  Old  diabetic 
urine  is  hence  especially  rich  in  such  acids.  The  higher  fatty  acids 
are  normally  not  observed  in  the  urine,  but  traces  may  appear  under 
certain  pathological  conditions. 

Isolation  and  Quantitative  Estimation. — To  isolate  the  volatile 
fattv  acids  the  collected  urine  of  twenty-four  hours  is  acidified  with 
phosphoric  acid  in  the  proportion  of  10  :  100,  and  distilled  in  a  cur- 
rent of  steam  so  long  as  the  distillate  shows  an  acid  reaction.  This 
is  then  neutralized,  evaporated  to  dryness,  and  the  residue  extracted 
with  alcohol.  Traces  of  sodium  chloride,  which  are  formed  on  the 
addition  of  the  alkali,  owing  to  the  presence  of  a  little  hydrochloric 
acid  that  has  passed  over,  remain  behind.  The  alcoholic  solution 
is  then  evaporated  to  dryness,  the  residue  is  dissolved  in  a  little 
water,  acidified  with  sulphuric  acid,  and  set  aside  in  the  cold,  when 
traces  of  benzoic  acid  are  precipitated.  The  filtrate  is  neutralized 
with  a  solution  of  sodium  carbonate  and  extracted  with  ether,  which 
removes  the  phenols.  The  solution  is  now  acidified  with  sul])huric 
acid  and  is  again  distilled  in  a  current  of  steam,  when  the  volatile 
fatty  acids  pass  over.  Their  presence  can  be  established  according 
to  the  common  methods  of  analysis.  To  estimate  the  amount,  the 
final  distillate  is  neutralized  with  barium  hydrate,  evaporated  to  dry- 
ness, and  the  residue  weighed.     This  weight  less  that  of  the  barium, 


THE  AROMATIC  OXY- ACIDS.  285 

which  is  in  combination,  and  which  can  be  determined  as  barium 
sulphate,  after  incineration  and  extraction  with  dihite  hydrochloric 
acid,  indicates  the  amount  of  the  fatty  acids  in  general. 

/3-Oxybutyric  Acid. 

This  acid  is  never  found  in  the  urine  under  normal  conditions. 
It  is  principally  met  with  in  the  severer  forms  of  diabetes,  when  it 
is  associated  with  the  presence  of  diacetic  acid  and  acetone.  It  may, 
however,  also  be  found  in  other  diseases,  as  in  the  continued  fevers, 
in  cachectic  conditions,  inanition,  etc.  As  a  general  rule  it  is  found 
in  combination  with  the  common  alkalies  of  the  blood,  but  when  it 
is  produced  in  especially  large  amounts  a  corresponding  quantity  of 
ammonia  is  furnished  by  the  body  to  effect  its  neutralization.  It 
may  happen,  however,  that  the  acid  formation  exceeds  that  of  am- 
monia, and  in  such  cases  the  free  acid  occurs  in  the  urine,  and  can 
also  be  demonstrated  in  the  blood  as  such.  Symptoms  of  acid  in- 
toxication then  exist,  and  it  is  noteworthy  that  in  such  cases  tiie 
amount  of  carbonic  acid  in  the  blood  has  been  found  markedly 
diminished,  showing  that  the  alkaline  salts  are  not  present  in  suffi- 
cient amount  to  remove  the  carbonic  acid  from  the  tissues. 

As  regards  the  origin  of  the  ^9-oxybutyric  acid,  there  is  evidence 
to  show  that  it  may  originate  from  the  albumins,  but  it  is  question- 
able whether  this  is  its  only  source.  Some  writers  claim  that  it  may 
also  originate  from  the  fats,  and  they  adduce  as  proof  the  observa- 
tion, that  the  amount  of  acetone  which,  as  seen  below,  is  a  deriva- 
tive of  /?-oxy butyric  acid,  is  increased  by  a  diet  rich  in  fats,  even 
though  there  may  be  no  increased  destruction  of  tissue-albumins. 
On  the  other  hand,  it  may  be  said  tliat  no  proof  has  been  offered 
to  show  that  an  increased  destruction  of  tissue-^dit  increases  the 
production  of  acetone,  and  it  is  quite  likely  that  the  increased 
elimination  on  a  diet  rich  in  fats  is  referable  to  an  increased  produc- 
tion in  the  alimentary  canal  as  the  result  of  bacterial  action,  and 
resultant  absorption  and  elimination  in  the  urine.  More  plausible 
is  the  supposed  origin  of  ^-oxybutyric  acid  from  the  carbohydrates. 
During  the  katalysis  of  the  latter,  and  notably  the  hexoses,  accord- 
ing to  Magnus-Levy,  lactic  acid  is  formed,  wliich  is  readily  decora- 
posed  into  formic  acid  and  acetic  aldehyde.  Through  a  condensation 
of  two  molecules  of  the  latter  /?-oxybutyric  aldehyde  would  then  be 
formed,  which  on  oxidation  yields  /9-oxybutyric  acid,  as  shown  by 
the  equations  : 

(1)  CfiHiA  =  2  CH3.CHOH.COOH 

(2)  CH3.CHOH.COOH  =  H.COOH  +  CH3.CHO 

(3)  CH3.CHO  +  CH3.CHO  =  CH3.CHOH.CH2.CHO 

(4)  CH3.CHOH.CH2.CHO  +  O  =  CH3.CHOH.CH2.COOH 

The  amount  of  oxybutyric  acid  which  may  occur  in  the  urine  is 
extremely  variable.     In  the  milder  cases  of  diabetes  it  is  usually 


286  THE   URINE. 

absent ;  in  tlie  sev^erer  forms,  liowcver,  large  quantities  may  be 
found,  and  Kiilz  reports  that  in  three  cases  a  daily  elimination  of 
67,  100,  and  226  grammes,  respectively,  ^vas  observed. 

The  chemical  relation  Avhich  exists  between  /9-oxybutyric  acid, 
diacetic  acid,  and  acetone  is  seen  from  the  equations  : 

(1)  CH3.CH(0H).CH,.C00H  +  O  =  (CH3.CO).CFl2.COOH  +  H^O. 

^-oxybutyric  acid.  Diacetic  acid. 

(2)  (CH,.CO)CH.,.COOH  =  CO{Cl%\  +  COj. 

Diacetic  acid.  Acetone. 

We  can  thus  readily  understand  that  in  certain  conditions  acetone 
may  be  found  in  the  urine  alone,  while  in  others  diacetic  acid,  and 
in  still  others  /?-oxybutyric  acid  may  be  present  as  well. 

On  boiling  /9-oxybutyric  acid  in  aqueous  solution  with  dilute 
mineral  acids,  a-crotonic  acid  results,  and  it  is  thus  apparent  that 
this  acid  is  also  found  in  the  distillate,  when  urine  containing  the 
first  is  distilled  with  sulphuric  acid.  Otherwise,  however,  it  does 
not  occur.  The  reaction  which  takes  place  may  be  represented  by 
the  equation  : 

CH,.CH(OH).CH2.COOH  ^  CHg.CH^^CHCOOH  +  H^O 

/3-oxybiityric  acid.  a-crotonic  acid. 

Test. — As  the  presence  of  oxybutvric  acid  presupposes  that  of 
diacetic  acid,  and  as  the  presence  of  the  latter  can  much  more 
readily  be  demonstrated  than  that  of  oxybutyric  acid,  a  test  in  this 
direction  should  always  precede  a  more  detailed  examination  (see 
below).  If  a  positiv^e  reaction  is  thus  obtained,  any  sugar  that  may 
be  present  is  removed  by  fermentation.  The  liquid  is  cleared  by 
adding  neutral  acetate  of  lead,  when  the  filtrate  is  examined  Avith 
the  polarimeter.  Should  Isevorotation  now  be  observed,  the  presence 
of  oxybutyric  acid  is  rendered  very  probable.  To  demonstrate  this 
beyond  a  doubt,  the  liquid  is  evaporated  to  a  syrup,  treated  with  an 
equal  volume  of  concentrated  sulphuric  acid  and  distilled,  without 
cooling.  In  this  manner  the  oxybutyric  acid  is  decomposed  with 
the  formation  of  a-crotonic  acid,  which  is  accordingly  found  in  the 
distillate.  If  this  is  present  in  larger  amounts,  it  crystallizes  out  in 
the  distillate,  when  this  is  strongly  cooled,  and  may  be  identified  by 
its  melting-point,  72°  C.  Should  smaller  amounts,  however,  be 
present,  crystallization  does  not  occur.  In  this  case  the  distillate  is 
extracted  with  ether  by  shaking.  Traces  of  benzoic  acid  and  the 
phenols  are  thus  likewise  extracted,  but  if  then  the  residue  of  the 
ethereal  solution  is  washed  with  water  other  impurities  are  removed, 
and  the  crotonic  acid  remains. 

Quantitative  Estimation  (according  to  Darmstaedter). — The  method 
is  based  on  the  decomposition  of  the  /9-oxybutyric  acid  with  the  for- 
mation of  a-crotonic  acid  and  the  estimation  of  the  latter.  100  c.c. 
of  urine  are  rendered  feebly  alkaline  with  sodium  carbonate  and 
evaporated  on  a  water-bath  almost  to  dryness.  With  the  aid  of 
150-200  c.c.  of  sulj)huric  acid  (50-55  per  cent.)  the  residue  is 
transferred  to  a  liter  flask,  which  is  closed  with  a  doubly  perforated 


THE  AROMATIC  OXY-ACIDS.  287 

stopper.  Through  the  one  aperture  a  drip-tube  passes,  while  a  bent 
glass  tube  passes  through  the  other  to  a  condenser.  Heat  is  applied, 
ut  first  mildly,  so  as  to  avoid  foaming  ;  then  vigorously.  Water  is 
allowed  to  enter  through  tlie  drip-tube  as  fast  as  the  distillate  passes 
over.  The  distillation  is  interrupted  when  from  300  to  350  c.c.  have 
been  obtained,  which  usually  takes  from  two  to  two  and  one-half 
hours.  The  distillate  is  extracted  two  or  three  times  with  ether. 
The  ether  is  distilled  oif,  the  residue  heated  for  a  few  minutes  on  a 
sand-bath  to  160°  C.  in  order  to  drive  off  any  fatty  acids  that  may 
be  present,  and  then  dissolved  on  cooling  with  50  c.c.  of  water. 
The  solution  is  filtered,  and  the  filter  washed  with  a  little  water. 
The  aqueous  solution  of  the  crotonic  acid  is  now  titrated  with  a 
decinormal  sodium  hydrate  solution,  using  phenolphtlialein  as  an 
indicator.  1  c.c.  of  the  soda  solution  corresponds  to  0.0086  gramme 
of  crotonic  acid.  The  corresponding  amount  of  oxybutyric  acid  is 
obtained  by  multiplying  by  1.21.  Sugar  does  not  interfere  with  the 
process. 

From  what  has  been  said  above,  it  is  clear  that  every  urine  which 
contains  /9-oxybutyric  acid  must  also  contain  diacetic  acid,  and  in 
extreme  cases  both  may  be  found  in  the  blood  as  such.  On  the  other 
hand,  it  will  also  be  understood  that  diacetic  acid  may  occur  in  the 
urine  in  the  absence  of /5-oxybutyric  acid,  and  this  is  indeed  more 
common. 

Diacetic  Acid. 

Tests. — As  diacetic  acid  is  rapidly  decomposed  on  standing,  it  is 
necessary  that  the  urine  should  be  as  fresh  as  possible,  when  it  is  to 
be  examined  in  this  direction.  To  this  end,  several  direct  tests  are 
available. 

Arnold's  Test. — This  test  is  the  most  reliable,  as  it  does  not 
respond  to  acetone  and  ^-oxybutyric  acid,  nor  to  the  common  anti- 
pyretics, salicylic  acid,  or  bile-pigment.  Highly  colored  urines, 
however,  should  first  be  filtered  through   animal  charcoal. 

Two  solutions  are  employed,  viz.,  a  1  per  cent,  solution  of  sodium 
nitrite,  and  a  solution  of  para-amido-acetophenon.  The  latter  is 
prepared  as  follows  :  1  gramme  of  the  acetophenon  is  dissolved  in 
from  80  to  100  c.c.  of  distilled  water,  and  treated  drop  by  drop 
with  hydrochloric  acid  until  the  solution,  which  at  first  is  yellow, 
becomes  entirely  colorless  ;  an  excess,  however,  should  be  carefully 
avoided.  Before  using,  the  two  solutions  are  mixed  in  the  propor- 
tion of  one  part  of  the  nitrite  to  two  of  the  acetophenon  solution. 
A  few  cubic  centimeters  of  the  urine  are  then  treated  with  an  equal 
volume  of  the  reagent,  and  a  few  drops  of  ammonia.  All  urines 
thus  give  a  more  or  less  well-marked  brownisli-red  color  on  shaking, 
and  in  the  presence  of  much  diacetic  acid  an  amorphous  precipitate 
of  the  same  color  is  formed.  If  now  a  small  amount  of  the  colored 
solution  is  treated  with  an  excess  of  concentrated  hydrochloric   acid 


288  THE   URINE. 

(10-12  o.c.  for  every  1    cc),  a  beautiful   purplish-violet  color  de- 
velops if  diacetic  acid  is  present. 

Gerhardt's  Test, — In  its  original  form  this  test  also  reacts 
with  the  common  antipyretics,  and  it  is  hence  necessary  to  isolate 
the  diacetic  acid  to  a  certain  degree.  To  this  end,  a  few  cubic  centim- 
eters of  the  urine  are  strongly  acidified  with  sulphuric  acid  and 
extracted  Avith  ether,  which  takes  up  the  acid.  The  extract  is  then 
shaken  with  a  few  cubic  centimeters  of  a  dilute  solution  of  the 
chloride  of  iron,  when  in  the  presence  of  diacetic  acid  the  aqueous 
layer  assnmes  a  violet  or  Bordeaux-red  color.  This,  however,  is  not 
permanent,  and  soon  fades  on  boiling  the  solution. 

Acetone. 

Traces  of  acetone,  varying  between  0.008  and  0.027  gramme  in 
the  twenty-four  hours  are  normally  found  in  the  urine.  Larger 
quantities  are  met  witii  if  the  carbohydrates  are  Avithdrav.  n  from  the 
diet,  and  in  such  cases  from  0.2  to  0.7  gramme  may  be  excreted 
after  the  sixth  day.  If  then  carbohydrates  are  again  ingested,  the 
elimination  of  the  acetone  rapidly  reaches  its  original  figure.  Gen- 
erally speaking,  small  amounts  of  carbohydrates  diminish  the  degree 
of  acetonuria,  while  larger  amounts  cause  the  acetone  to  disappear 
altogether.  The  ingestion  of  fats,  on  the  other  hand,  is  without 
effect  in  this  respect. 

Most  authors  regard  the  albumins  as  the  source  of  the  acetone, 
and  as  a  matter  of  fact  the  elimination  of  acetone  is  most  marked 
during  starvation  and  with  an  exclusive  albuminous  diet  not  quite 
sufficient  to  cover  the  loss  of  tissue-albumins.  However,  it  has  been 
observed  that  notable  quantities  of  acetone  may  appear  in  the  urine 
while  there  is  a  gain  of  tissue-albumins,  and  that  the  acetonuria 
may  still  be  caused  to  disappear  by  the  administration  of  carbohy- 
drates, even  though  the  amount  of  albumin  may  be  insufficient. 
Hirschfeld  concludes  that  the  formation  of  acetone  is  not  the  result 
of  the  destruction  of  tissue-albumin  during  starvation,  but  owing  to 
the  absence  of  carbohydrates.  It  is  supposed  that  in  some  manner 
the  decomposition  of  the  carbohydrates  leads  to  a  destruction  of  the 
acetone,  which  in  turn  is  derived  from  the  albumins.  These  obser- 
vations, however,  apply  only  to  the  human  being.  In  animals  con- 
ditions seem  to  be  somewhat  different,  and  in  the  dog,  for  example, 
the  amount  of  acetone  in  the  urine  is  directly  proportionate  to  the 
amount  of  nitrogen  eliminated,  and  is  altogether  uninfluenced  by  the 
simultaneous  administration  of  other  foods. 

Other  observers,  like  Magnus-Levy  and  Geelmuyden,  regard  the 
fat  as  the  principal  source  of  the  acetone,  and  Schwarz  even  goes  so 
far  as  to  denv  tlie  origin  from  albumins  altogether.  Asa  matter  of 
fact,  acetone  can  be  readily  derived  from  fatty  acids,  and  until  very 
recentlv  an  absolute  proof  of  the  possible  origin  of  acetone  by  hy- 
drolvsis  or  oxidation  from  albumin  had  not  been  furnished  in  the 


THE  AROMATIC  OXY-ACIDS.  289 

laboratory.  Blumenthal  and  Neuberg,  however,  have  now  shown 
that  this  is  possible  by  oxidizing  gelatin  with  hydrogen  peroxide  in 
the  presence  of  an  iron  salt. 

Acetonuria  is  essentially  a  pathological  phenomenon,  and  is  ob- 
served in  most  pronounced  form  in  severe  cases  of  diabetes,  in 
which,  as  I  have  stated,  it  is  frequently  met  with  in  association 
with  ^3-oxybutyric  acid  and  diacetic  acid.  Like  diacetic  acid,  how- 
ever, it  may  occur  in  the  absence  of  oxybutyric  acid,  and  in  the 
milder  forms  of  diabetes,  as  also  under  normal  conditions,  it  may  be 
present  alone.  But  while  its  import  is  generally  the  same  as  that 
of  its  immediate  antecedents,  it  appears  that  it  may  originate  also  in 
the  gastro-intestinal  tract  as  the  result  of  some  abnormal  form  of 
albuminous  putrefaction,  and  it  is  possible,  indeed,  that  the  so- 
called  asthma-acetouicura  may  be  of  this  origin.  That  small 
amounts  are  formed  also  during  the  hydrolytic  decomposition  of  the 
albumins  with  boiling  mineral  acids  or  the  caustic  alkalies,  has  been 
mentioned,  v.  Jaksch,  further,  has  shown  that  acetone  may  be 
formed  as  a  by-product  during  the  process  of  lactic  acid  fermenta- 
tion, and  it  has  been  suggested  that  the  small  amounts  which  are 
normally  met  with  in  the  urine  may  originate  in  this  manner.  At 
present,  however,  we  are  not  in  a  position  to  speak  authoritatively 
of  the  origin  of  these  normal  traces,  and,  pathologically  at  least,  we 
can  thus  far  acknowledge  but  one  source  of  the  acetone,  viz.,  the 
albumins  of  the  body-tissues,  and  secondarily  perhaps  the  circulat- 
ing albumins. 

Tests. — Should  diacetic  acid  be  demonstrated  in  the  urine,  the 
simultaneous  presence  of  acetone  may  be  directly  inferred.  If  this 
is  not  the  case,  it  is  best  to  distill  from  250  to  500  c.c.  of  the  urine, 
after  the  addition  of  a  small  amount  of  phosphoric  acid,  and  to 
apply  the  following  tests  to  the  first  15  or  30  c,c.  of  the  distillate 
that  has  passed  over. 

Legal's  Test. — A  few  cubic  centimeters  of  the  distillate  are 
treated  ^vith  several  drops  of  a  freshly  ]5repared,  concentrated  solu- 
tion of  sodium  nitroprusside,  and  a  small  amount  of  a  dilute  solu- 
tion of  sodium  hydrate.  In  the  presence  of  acetone  a  red  color 
develops,  which  rapidly  fades  however,  but  is  replaced  by  a  beau- 
tiful carmin  or  purple  red  if  the  solution  is  treated  with  acetic 
acid  in  excess ;  on  standing,  this  turns  to  a  bluish  violet, 

Lieben's  Test. — On  adding  a  few  drops  of  a  dilute  solution  of 
iodopotassic  iodide  to  a  small  amount  of  the  distillate  that  has  been 
rendered  alkaline  with  sodium  hydrate  solution,  a  precipitate  of 
iodoform  develops  in  the  presence  of  acetone,  which  may  readily  be 
recognized  by  its  odor  on  warming  the  mixture.  This  test,  how- 
ever, is  not  conclusive,  as  other  substances,  such  as  alcohol,  give  the 
same  reaction. 

Gunning's  Test,  as  modified  by  Salkowski. — A  small 
amount  of  the  distillate  is  treated  drop  by  drop  with  freshly  pre- 
cipitated and  well-washed  mercuric  oxide  in   alcoholic    suspension 

19 


290  THE   URINE. 

until  a  portion  of  the  oxide  remains  undissolved.  The  liquid  is 
then  filtered,  and  the  filtrate  supeq)osed  with  a  solution  of  ammo- 
nium sulphide,  when  in  the  presence  of  acetone  the  zone  of  contact 
assumes  a  grayish-black  color,  owing  to  the  formation  of  mercuric 
sulphide. 

Quantitative  Estimation. — The  quantitative  estimation  of  acetone 
is  best  conducted  according  to  the  method  of  Messinger,  as  modified 
by  Huppert.  It  is  based  u]ion  the  j^rinciple  underlying  Lieben's 
test,  viz.,  the  formation  of  iodoform  when  a  dilute  solution  of  iodo- 
potassic  iodide  is  added  to  an  alkaline  solution  of  acetone.  By 
determining  the  amount  of  iodine  which  is  consumed  in  this  reac- 
tion the  corresponding  amount  of  acetone  can  then  be  calculated. 

One  hundred  c.c.  of  urine,  or  less  if  much  acetone  is  present,  as 
determined  by  Legal's  test  applied  directly  to  the  urine,  are  treated 
with  2  c.c.  of  a  50  per  cent,  solution  of  acetic  acid  and  distilled 
until  all  the  acetone  has  passed  over.  The  distillate  is  received  in 
a  bulb-tube  containing  water.  The  solution  which  thus  results  is 
treated  with  1  c.c.  of  a  12  per  cent,  solution  of  sulphuric  acid  and 
redistilled.  The  second  distillate  is  free  from  phenols.  To  it  a 
carefullv  measured  quantity  of  a  one-tenth  normal  solution  of  iodine 
is  added  (10  c.c.  for  every  100  c.c.  of  urine),  together  with  a  50 
per  cent,  solution  of  sodium  hydrate,  until  the  iodoform  separates 
out.  After  shaking,  the  mixture  is  set  aside  for  a  few  minutes,  and 
then  acidified  with  concentrated  hydrochloric  acid.  If  iodine  is 
present  in  excess,  a  brown  color  thus  develops.  This  excess  is  then 
titrated  with  a  decinormal  solution  of  sodium  thiosulphate,  using 
starch  solution  as  a  final  indicator.  The  number  of  cubic  centim- 
eters emyloyed  in  this  titration  is  deducted  from  the  amount  of 
the  iodine  solution  added.  The  difference  multiplied  by  0.967  then 
indicates  the  amount  of  acetone  in  the  100  c.c.  of  urine,  in  milli- 
grammes. 

Lactic  Acid. 

As  I  have  shown,  there  is  reason  to  believe  that  the  greater  por- 
tion of  the  nitrogen  which  is  set  free  in  the  katabolism  of  the 
various  tissues  appears  in  the  form  of  the  ammonium  salt  of  lactic 
acid  and  is  transformed  into  urea  in  the  liv^er.  Normally,  indeed, 
lactic  acid  is  not  found  in  the  urine.  It  occurs,  however,  when  the 
further  transformation  of  the  ammonium  salt  is  impeded,  and  is 
hence  met  with  in  various  diseases  of  the  liver  which  are  associated 
with  an  extensive  destruction  of  the  hepatic  parenchyma,  as  also  in 
conditions  in  which  the  oxidation-processes  of  the  body  are  im])aired 
in  general.  It  is  thus  notably  met  with  in  acute  yellow  atrophy,  in 
poisoning  with  phosphorus  and  carbon  monoxide,  in  long-continued 
anaemic  conditions,  etc.  Smaller  amounts  have  been  found  in  soldiers 
after  marches,  and  in  epileptic  patients  after  severe  seizures. 

Isolation. — To  isolate  the  substance  from  the  urine  the  following 
method  may  be  employed  as  suggested  by  Araki : 


THE  AROMATIC  OXY- ACIDS.  291 

The  collected  urine  of  twenty-four  hours  is  evaporated  to 
about  50  or  60  c.c,  treated  with  ten  times  as  much  of  95  per  cent, 
alcohol,  and  set  aside  for  twelve  hours.  It  is  then  filtered  and  freed 
from  the  alcohol  by  distillation.  The  residual  fluid  is  acidified  with 
phosphoric  acid,  and  repeatedly  extracted  with  five  times  its  volume 
of  ether.  The  ethereal  extract  is  evaporated  and  the  remaining 
yellow  syrup  dissolved  in  a  little  water.  Any  hippuric  acid  which 
may  be  present  thus  separates  out  and  is  filtered  off.  The  filtrate  is 
now  treated  with  pure  lead  carbonate  in  substance,  heated  on  a 
water-bath  for  thirty  minutes,  and  filtered  on  cooling.  From  the 
filtrate  the  lead  is  removed  by  means  of  hydrogen  sulphide,  and  the 
excess  of  the  latter  by  gently  warming  on  a  water-bath.  The 
fluid  is  then  concentrated  to  a  thick  syrup  and  extracted  with  ether. 
The  ethereal  solution  is  evaporated  and  the  residue  boiled  for  some 
time  with  water  and  an  excess  of  zinc  carbonate.  The  mixture 
is  filtered  while  hot,  concentrated  to  a  small  volume,  and  then  set 
aside  in  the  cold  after  adding  a  little  alcohol.  The  zinc  salts  of 
both  paralactic  acid  and  the  common  optically  inactive  lactic  acid 
which  may  also  be  present  in  traces,  then  crystallize  out.  They 
can  be  separated  from  each  other  by  treating  with  absolute  alcohol, 
in  which  the  latter  is  insoluble.  It  must  be  noted  that  the  solu- 
bility of  the  paralactate  is  also  slight  (1  :  1100),  so  that  it  is 
necessary  to  add  a  large  amount  of  alcohol.  In  order  to  prevent 
confusion  with  the  aromatic  oxy-acids  of  the  urine,  the  lactate 
crystals  should  now  be  further  identified,  which  is  most  conveniently 
done  by  estimating  the  water  of  crystallization.  The  paralactate 
contains  two  molecules  of  this,  which  escapes  at  105°  C,  and  at  this 
temperature  the  weight  of  the  crystals  should  therefore  diminish  12.9 
per  cent.  The  salt,  moreover,  like  its  acid,  is  Isevorotatory,  while 
the  common  lactate  is  optically  inactive. 

Leucin  and  Tyrosin. 

Leucin  and  tyrosin,  according  to  some  observers,  are  normally 
present  in  the  urine  in  traces.  By  others  this  is  denied,  and  I  must 
admit  that  I  have  never  found  either  of  the  substances  under  normal 
conditions.  In  certain  diseases  of  the  liver,  however,  in  which  exten- 
sive destruction  of  the  hepatic  cells  is  going  on,  both  may  be  found. 
But  it  is  noteworthy  that  while  in  acute  yellow  atrophy  this  is  a 
common  occurrence  after  the  first  week  of  the  disease,  in  acute  phos- 
phorus poisoning  they  are  usually  not  found.  Thus  far  we  have 
no  adequate  explanation  to  offer  for  this  difference,  and  we  are  in 
ignorance,  moreover,  of  the  origin  of  the  bodies  in  question  in  the 
former  disease.  We  have  seen  that  as  a  general  rule  at  least  the 
greater  portion  of  the  tissue  nitrogen  which  is  set  free  during  the 
process  of  metabolism  is  carried  to  the  liver  in  the  form  of  ammo- 
nium lactate,  and  there  is  no  evidence  to  show  that  this  may  be 
transformed  into  either  leucin  or  tyrosin.     We  see,  in  fact,  tiiat  in 


292  THE   URINE. 

extensive  hepatic  disease  ammonium  lactate  appears  in  the  urine. 
Whether  or  not  in  acute  yeUow  atrophy  leucin  and  tyrosin  are  also 
set  free  in  the  tissues  in  general,  we  do  not  know.  In  the  liver,  it  is 
true,  both  are  then  met  wdth  iu  large  amount,  but  we  may  readily 
suppose  that  the  substances  may  have  originated  here  directly,  and 
possibly  as  a  result  of  some  fermentative  action.  This,  indeed, 
appears  the  most  likely  explanation. 

Isolation. — If  leucin  and  tyrosin  are  present  in  the  urine  in  small 
amounts,  they  are  held  in  solution.  In  the  presence  of  larger 
amounts  the  tyrosin  may  separate  out,  and  can  then  be  isolated  from 
the  sediment  and  identified  as  described.  Leucin,  however,  is  rarely 
found  in  this  manner,  and  remains  in  solution  even  though  very 
large  quantities  are  eliminated. 

To  demonstrate  both  w4ien  they  are  held  in  solution,  it  is  some- 
times only  necessary  to  concentrate  a  small  amount  of  urine  on  a 
water-bath  and  to  examine  the  residual  syrup  with  the  microscope. 
Otherwise  it  is  advisable  to  precipitate  the  collected  urine  of 
twenty-four  hours,  after  removing  any  albumin  that  may  be  present, 
with  basic  lead  acetate.  The  filtrate  is  then  freed  from  lead  with 
hydrogen  sulphide,  evaporated  to  a  thick  syrup,  and  set  aside  for 
crystallization.  Tyrosin  and  leucin  can  then  be  demonstrated  by  a 
microscopical  examination  and  identified  in  the  usual  manner  (see 
page  207). 

THE  NEUTRAL  SULPHUR  BODIES  OF  THE  URINE. 

In  the  section  on  the  mineral  constituents  of  the  urine  I  pointed 
out  that  the  greater  portion  of  the  sulphur  which  is  set  free  during 
the  metabolism  of  the  nitrogenous  constituents  of  the  body  is  elimi- 
nated in  the  urine  in  a  completely  oxidized  form.  A  much  smaller 
fraction,  however,  escapes  oxidation,  and  appears  in  the  urine  as 
so-called  neutral  sulphur.  Normally  this  constitutes  about  12-15 
per  cent,  of  the  total  amount. 

The  neutral  sulphur  bodies  which  have  been  described  in  the  urine 
of  man  under  normal  conditions  are  certain  sulphocyanides,  the  so- 
called  oxyproteinic  acid  and  alloxyproteinic  acid  of  Bondzynski  and 
Gotlieb  and  Panek  respectively,  the  uroproteic  acid  of  Cloetta,  and 
the  uroferric  acid  of  Tiiiele.  The  sulphocyanides  in  question  are 
probably  derived  from  the  saliva  and  the  gastric  juice,  where  they 
are  normally  found  in  traces.  The  two  proteinic  acids,  as  also  the 
uroproteic  acid,  have  probably  not  been  obtained  in  pure  form.  The 
first  is  said  to  have  the  formula  C^^H^^Ni^SO,,,  the  last  C,;,;H,iyN2oSO,^. 
The  uroferric  acid,  according  to  Thiele,  has  the  formula  C3,H.,p,Nj,SO,9 ; 
it  contains  3.46  per  cent,  of  sulphur,  of  which  about  one-half  can  be 
split  off  on  prolonged  boiling  with  hydrochloric  acid  or  sulphuric 
acid.  In  this  respect  therefore  it  shows  the  behavior  of  a  conjugate 
sulphate.  In  addition,  however,  it  contains  sulphur,  which  cannot 
be  split  off  even  on  prolonged  boiling  with  alkaline  solution  of 
acetate  of  lead.     Its  amount  is  quite  small. 


THE  AROMATIC  OXY- ACIDS.  293 

In  the  urine  of  cats,  and  less  constantly  of  dogs,  traces  of  tliio- 
sulphates  are  found,  while  in  man  they  are  normally  absent.  They 
have  been  once  found  in  a  case  of  typhoid  feyer.  Cystein  and 
ethyl  sulphide  are  constant  constituents  of  the  urine  of  dogs. 

Whether  or  not  taurocarbaminic  acid  is  constantly  present  in 
human  urine  has  not  been  ascertained.  I  have  shown,  however, 
that  to  a  certain  extent  at  least  taurin  is  eliminated  in  this  form 
when  given  by  the  mouth.  In  cases  of  obstructive  jaundice,  more- 
over, or  after  ligation  of  the  common  duct  in  dogs,  the  neutral 
sulphur  may  increase  to  40  per  cent,  of  the  total  amount,  and  it  is 
known  that  in  such  cases  taurocarbaminic  acid  is  constantly  present. 
Its  formation  may  be  represented  by  the  equation  : 

S02<  +  co<        =  co< 

\0H  ^NHj  \NH.C2H4.S02.0.NH4. 

Taurin.  Urea.  Ammonium  taurocarbamate. 

In  all  probability  this  synthesis  is  effected  in  the  kidneys.  In 
rabbits,  on  the  other  hand,  taurin  is  largely  oxidized  to  sulphuric 
acid,  while  a   small  portion  appears  as  thiosulphuric  acid. 

Cystein. — On  feeding  dogs  with  halogen  benzols,  peculiar  products 
appear  in  the  urine,  which  contain  both  sul|)hur  and  nitrogen,  and 
which  are  apparently  united  with  glucuronic  acid.  Baumann  and 
Preusse  have  termed  these  mercapturic  acids.  FolloM'ing  the  ad- 
ministration of  monobrom  benzol  they  could  isolate  a  brom-phenyl 
mercapturic  acid  of  the  composition  CnHj^BrSXOg,  M-hich  on  hydrol- 
ysis yielded  acetic  acid  and  a  substance  of  the  formula  C9HioBrXS02 
according  to  the  equation  : 

CiiHijBiSXOj  -  H.,0  =  CH3.COOH  -f  CgHioBrXSO. 

This  product  they  subsequently  identified  as  brom-phenyl  cystein, 
and  the  proof  had  thus  been  furnislied  that  cy.stein  is  normally 
produced  in  the  sulphur  metabolism,  of  the  dog,  at  any  rate.  It  is 
identical  with  the  cystein  which  can  be  obtained  from  the  albumi- 
nous cystin  on  reduction,  and  the  elimination  of  mercapturic  acid 
can  hence  be  regarded  as  an  experimental  cystinuria. 

The  amount  of  cystein  which  is  normally  present  in  the  urine 
probably  does  not  exceed  0.015  gramme  in  the  twenty-four  hours. 
Larger  quantities  are  found  under  pathological  conditions,  as  in 
phosphorus  poisoning,  but  on  the  whole  its  elimination  in  disease 
has  received  little  attention. 

Cystin. — As  has  been  shown,  cystin  is  a  constant  decomposition- 
product  of  all  albumins  and  represents  one  of  the  primary  radicles 
of  the  original  molecule.  Under  normal  conditions  it  is  not  found 
in  the  urine.  As  in  the  case  of  alkapton,  its  presence  re])resents 
the  existence  of  a  distinct  metabolic  anomaly,  of  the  true  nature  of 
which,  however,  nothing  is  known.     Very  curiously  its  presence  in 


294  THE   URINE. 

the  urine  mav  be  associated  M'itli  the  simultaneous  presence  of  cer- 
tain (liamins,  viz.,  cadaverin  and  })utrescin.  As  these  were  formerly 
reo^arded  as  specific  products  of  bacterial  activity,  their  presence 
was  brought  into  a  supposed  causal  relationship  to  thatof  cystinuria. 
In  the  light  of  more  modern  investigations,  however,  we  may  assume 
that  the  diaminuria  in  these  cases  is  in  all  likelihood  also  the  expres- 
sion of  a  metabolic  anomaly,  and  like  the  cystinuria  of  histogenic 
origin. 

Outside  of  the  urine  cystin  has  been  encountered,  as  such,  in  a 
few  instances  only.  Cloetta  claims  to  have  found  it  in  the  kidneys 
of  the  ox,  Scherer  in  the  liver  of  a  patient  dead  with  typhoid  fever, 
and  Drechsel  isolated  the  body  from  the  liver  of  a  horse  and  a 
porpoise.  More  recently  Abderhalden  has  described  the  case  of  a 
child  which  died  at  the  age  of  twenty-one  months  and  a  half,  and 
in  which  post  mortem  the  various  organs,  and  notably  the  liver  and 
spleen,  were  found  incrustated  \v\i\\  cystin  crystals. 

Clinically  cystin  is  of  interest  as  its  elimination  in  the  urine 
favors  the  formation  of  cystin  concretions. 

The  amount  which  may  be  met  with  is  extremely  variable.  On 
some  days  traces  only  are  found,  while  on  others  the  elimination 
may  amount  to  a  gramme  or  more.  The  neutral  sulphur  is  corre- 
spondingly increased  (to  60  per  cent,  of  the  total  and  even  higher). 
Very  commonly  the  cystin  separates  out  in  crystals  shortly  after 
being  voided,  but  sometimes  it  is  necessary  to  acidify  the  urine 
strongly  with  an  excess  of  acetic  acid.  On  decomposition  such 
urines  develop  a  strong  odor  of  hydrogen  sulphide. 

Properties. — Structurally  cystin  is  a-dianiido-/5'-dithio-dilactic 
acid ;  it  is  the  disulphide  of  /3-cystem  and  results  from  this  on 
oxidation  according  to  the  equation  : 

CH.,.SH  CH.,.S S.CH2 

I     ■  I     "  I 

2CH.NH2    +   O   =   CH.NH2  CH.XH2   +   H2O 

I  '  I 

COOH  COOH  (X)OH 

Cystein.  Cystin. 

On  oxidation  with  bromine  it  is  possible  to  transform  the  loosely 
combined  sulphur  into  oxidized  sulphur  with  the  production  of 
cysteinic  acid,  which  apparently  represents  the  sulpho-acid  of  cystein. 
Its  probable  formula  is 

CH,.SO.pH 

I 
CH.NH2 

I 
COOH 

Through  loss  of  COo  this  then  gives  rise  to  taurin  (which  see). 

Two  varieties  of  cystin  exist,  of  which  one  is  Isevorotatory  and 
the  other  dextrorotatory.  The  variety  which  is  found  in  the  urine 
is  Isevorotatorv.      The   substance  usuallv  crvstallizes    in    colorless 


THE  AROMATIC  OXY-ACIDS.  295 

hexagonal  platelets  which  are  very  characteristic  in  appearance. 
But  it  may  also  separate  out  in  needles,  whicli  diifer  from  those  of 
tyrosin  in  the  fact  that  they  appear  more  highly  refractive  under 
the  microscope  and  present  obliquely  cut  ends.  On  recrystallization 
from  a  10  per  cent,  solution  of  ammonia  these  needle-like  crystals 
disappear  and  are  replaced  by  hexagonal  platelets. 

Cystin  is  soluble  in  solutions  of  the  alkaline  hydrates,  in  ammonia, 
and  the  mineral  acids.  In  water,  alcohol,  ether,  and  acetic  acid  it 
is  insoluble,  as  also  in  solutions  of  ammonium  carbonate ;  for  this 
reason  the  cystin  is  apt  to  crystallize  out  from  decomposing  urines, 
when  previously  it  has  been  present  in  solution  only. 

On  heating  cystin  on  platinum  foil  it  does  not  melt,  but  ignites 
and  burns  with  a  bluish-green  flame ;  at  the  same  time  a  peculiar, 
penetrating  odor  develops.  It  does  not  give  the  murexid  reaction. 
When  boiled  with  caustic  alkali  it  is  decomposed  and  the  sulphur 
liberated  as  a  sulphide.  With  benzoyl  chloride,  in  the  presence  of 
an  excess  of  caustic  alkali,  it  forms  benzoyl-cystin,  and  is  thus  pre- 
cipitated as  a  sodium  salt  in  the  form  of  fine  lustrous  platelets, 
which  are  readily  soluble  in  water,  but  insoluble  in  solutions  of  the 
caustic  alkalies.  Upon  the  addition  of  an  acid  to  such  a  solution, 
benzoyl-cystin  separates  out  as  such.  It  is  soluble  in  alcohol  and 
alcohol-ether,  slightly  so  in  pure  ether,  and  almost  insoluble  in 
water.  Its  needle-like  crystals  melt  at  156°-158°  C.  The  forma- 
tion of  benzoyl-cystin  may  be  expressed  by  the  equation  : 

CH,.S S.CH,  CH2.S S.CH2 

I'll  I 

2C6H5.COCI+CH.NH2        CH.NH2=CH.NH.C6H5CO  CglL.CO.NH.CH     +  2HC1 

III  I 

COOH  COOH       COOH  COOH 

Cystin.  Benzoyl  cystin. 

On  boiling  with  concentrated  hydrochloric  acid  benzoyl-cystin  is 
decomposed  with  the  formation  of  benzoic  acid  and  cystin. 

On  shaking  cystin  in  alkaline  solution  with  /9-naphthalin-sul- 
phochloride  (dissolved  in  ether)  and  subsequently  acidifying  with 
hydrochloric  acid  (after  removal  of  the  ether)  a  precipitate  of 
^-naphthalin-sulphocystin  is  formed,  of  the  composition  C^oHj^S^NjOg. 
This  dissolves  with  difficulty  in  water  and  cold  absolute  alcohol, 
but  readily  in  hot  absolute  alcohol ;  from  the  latter  the  substance 
crystallizes  out  in  flat,  partly  bent  needles,  which  at  215°  C.  (uncor- 
rected) decompose  with  the  formation  of  a  brown  oily  material. 

Isolation  of  Cystin  from  the  Urine  and  Quantitative  Estimation. — 
To  this  end  Abderhalden  has  recommended  the  following  method, 
based  on  the  principle  just  outlined.  The  entire  amount  of  urine 
of  twenty-four  hours  is  filtered  and  the  residue  washed  with 
ammonia.  The  filtered  urine  and  tlie  ammonia  washings  are 
united.  500  c.c.  (best  after  previous  concentration  in  the  vacuum 
at  40°  C.)  are  then  treated  with  4  c.c.  of  normal  sodium  hydrate 
solution  and  then  shaken  for  six  to  eight  hours  with  an  ethereal 


296  THE   URINE. 

solution  of  4  grammes  of  /9-naphthalin-sulphocliloride.  At  in- 
tervals of  one  and  a  half  hours  3  c.c.  of  tlie  alkali  solution  are 
further  added.  At  the  expiration  of  the  shaking  the  ether  layer 
is  removed  and  the  aqueous  solution  oversaturated  with  hydro- 
chloric acid.  The  resulting  precipitate  is  filtered  off  and  after 
decolorization  with  animal  charcoal  crystallized  from  hot  absolute 
alcohol.  The  crystals  are  dried  at  100°  C  and  weighed.  1  gramme 
of  the  compound  corresponds  to  0.35  gramme  of  cystin. 

Normal  urine  gives  no  precipitate  with  /^-naphthalin-sulphochlo- 
ride,  or  a  slight  turbidity  only. 

Preparation  of  Cystin  from  Albumins. — For  purjioses  of  study 
cystin  is  most  conveniently  obtained  from  human  hair  as  follows : 
500  grammes  of  hair  are  boiled  for  four  hours  with  1500  c.c.  of 
hydrochloric  acid  (specific  gravity  1.19).  On  cooling,  concentrated 
sodium  hydrate  solution  is  carefully  added  until  the  reaction  is 
nearly  neutral  (not  alkaline).  Animal  charcoal  is  added  in  excess ; 
the  mixture  is  boiled  for  about  three-quarters  of  an  hour  and 
filtered  while  hot.  On  cooling,  impure  cystin  separates  out.  This 
is  collected  on  a  filter,  dissolved  in  hot  dilute  ammonia,  and  repre- 
cipitated  by  the  addition  of  glacial  acetic  acid.  The  resulting  pre- 
cipitate is  again  dissolved  in  a  small  amount  of  hot  dilute  ammcjuia, 
the  solution  is  filtered,  when  on  spontaneous  evaporation  of  the 
ammonia  the  cystin  separates  out  in  rosettes  composed  of  the  typical 
hexagonal  platelets.  These  can  be  further  purified  by  a  repetition 
of  the  precipitation  of  the  ammoniacal  solution  with  glacial  acetic 
acid.  The  crystals  are  finally  washed  with  water,  alcohol,  and 
ether. 

Quantitative  Estimation  of  the  Neutral  Sulphur. — In  one  portion 
of  the  urine  the  oxidized  sulphur,  viz.,  the  mineral  and  the  conju- 
gate sulphur,  is  estimated  as  previously  described  (page  238).  In  a 
second  portion  the  total  sulphur  is  determined  as  follows  (the  differ- 
ence between  the  two  results  indicates  the  amount  of  neutral 
sulphur) : 

Method  of  Hohnel,  Glaser,  y.  Asboth  (modified  by 
Mo<lrakowski). — 1-2  grammes  of  sodium  peroxide  are  placed  in 
a  nickel  dish,  and  covered  with  50  c.c.  of  urine,  which  is  added 
from  a  pipette  drop  by  drop.  The  fluid  is  evaporated  on  a  water- 
bath  to  a  syrup,  and  is  further  treated  with  2-3  grammes  of  the 
Dcroxide,  which  is  added  slowly  and  carefully,  Avhile  stirring.  As 
soon  as  the  reaction,  wdiich  at  first  is  fairly  vigorous,  becomes 
calmer,  the  dish  is  removed  from  the  water-bath  and  heated  with  a 
small  alcohol  lamp,  if  necessary  adding  from  1  to  3  grammes  of 
peroxide  more.  The  mass  now  apjiears  as  a  brown  syrup  and  finally 
becomes  thick.  Tliis  ends  the  reaction.  On  cooling  the  fusion  is 
dissolved  in  hot  water ;  the  solution  is  filtered  and  feebly  acidified 
with  hydrochloric  acid.  Barium  chloride  is  then  added  and  the 
process  continued  as  described  elsewhere  (page  238). 


THE  CARBOHYDRATES.  297 

THE    CARBOHYDRATES. 

The  carbohydrates  which  may  be  found  in  the  urine  comprise 
glucose,  Iffivulose,  laiose,  maltose,  lactose,  dextrin,  animal  gum,  and 
certain  pentoses.  Of  these,  traces  of  glucose,  dextrin,  animal  gum, 
and  possibly  also  pentoses,  may  be  found  at  all  times.  Their 
amount,  however,  is  normally  so  small  that  their  presence  cannot  be 
recognized  by  the  common  tests.  Larger  amounts  of  carbohydrates 
are  found  in  health  only  during  the  puerperal  state,  and  in  the 
course  of  lactation,  when  lactose  is  commonly  present.  Otherwise 
the  elimination  of  sugar  in  amounts  which  can  be  demonstrated  by 
the  ordinary  tests  must  be  regarded  as  abnormal. 

Glucose. — As  I  have  indicated,  glucose  appears  in  the  urine 
whenever  its  amount  in  the  blood  exceeds  3  pro  mille.  This, 
however,  occurs  only  under  abnormal  conditions,  and  in  the  pres- 
ence of  small  amounts  the  kidneys  are  manifestly  capable  of 
preventing  its  passage  into  the  urine.  Under  certain  conditions, 
however,  this  power  is  apparently  lost,  and  we  find,  as  a  matter 
of  fact,  that  following  the  administration  of  phlorhizin  glucosuria 
occurs,  altliough  the  percentage  of  sugar  is  not  increased  in  the 
blood.  Whether  or  not  such  an  insufficiency  on  the  part  of  the 
kidneys  may  also  occur  spontaneously  we  do  not  know.  As  a  gen- 
eral rule,  however,  glucosuria  is  associated  with  a  hypergluchsemia. 
This  may  result  if  unduly  large  amounts  of  sugar  reach  the  liver, 
so  that  the  organ  is  incapable  of  transforming  the  entire  quantity 
into  glycogen,  and  I  have  pointed  out  that  the  functional  capacity 
of  the  liver  in  this  respect  is  of  a  much  lower  order  than  the 
ability  of  the  intestinal  epithelium  to  transform  polysaccharides 
and  disaccharides  into  glucose.  The  extent  to  which  the  liver 
can  normally  transform  glucose  into  glycogen  seems  to  vary 
with  different  individuals.  Generally,  glucosuria  occurs  when 
the  amount  of  sugar  exceeds  200  grammes.  There  are  many 
individuals,  however,  in  which  this  occurs  following  the  admin- 
istration of  only  150  grammes,  and  there  are  others  in  which  the 
ingestion  of  250  grammes  does  not  cause  glucose  to  appear  in 
the  urine.  Glucosuria  following  the  ingestion  of  100  grammes  of 
grape-sugar  is  now  regarded  as  abnormal,  and  there  is  reason  to 
believe  that  the  hepatic  insufficiency  thus  manifested  may  be  of  the 
type  of  a  mild  form  of  diabetes.  The  amount  of  sngar  which  then 
appears  in  the  urine  rarely  exceeds  3  per  cent.  The  glucosuria, 
moreover,  is  only  temporary,  and  disappears  as  soon  as  the  ingestion 
of  sugar  (viz.,  starches)  is  diminished.  Between  this  form  of  glu- 
cosuria and  the  common  form  of  diabetes,  in  which  practically  no 
sugar  can  be  utilized  by  the  body,  but  in  which  the  elimination 
ceases  as  soon  as  carbohydrates  are  withdrawn  from  the  diet,  all 
gradations  may  occur.  Tliese  forms  are  now  generally  regarded  as 
referable  to  a  hepatic  insufficiency  of  whatever  origin.  Quite  differ- 
ent from  diabetes  of  this  character,  on  the  other  hand,  is  the  type 


298  THE   URINE. 

in  M'hicli  the  gluoosuria  continues  although  no  sugars  are  ingested. 
In  such  cases  a  hepatic  insufficiency  need  not  necessarily  exist, 
and  there  is  evidence  to  show  that  in  these  forms  the  formation 
of  glycogen  may  still  occur.  We  must  therefore  assume  that 
other  organs  are  primarily  involved,  and  there  is  every  reason  to 
suppose  that  the  metabolism  of  muscle-tissue  is  here  princij)allv 
at  fault,  and  that  the  tissue  has  lost  the  power  of  decomposing 
the  sugar  which  reaches  it  from  the  liver.  As  a  consequence, 
increased  destruction  of  muscle-tissue  occurs,  as  the  inability  on 
the  part  of  these  structures  to  decompose  sugar  amounts  to  the 
same  as  though  no  sugar  were  present  at  all.  The  b(^dy  therefore 
furnishes  glucose  from  its  albumins  to  supply  the  apparent  deficit, 
and  thus  further  increases  the  hypergluca^mia  and  the  resulting 
glucosuria.  We  accordingly  find  that  even  though  the  carbohy- 
drates have  been  withdrawn  from  the  food  sugar  still  appears  in 
the  urine.  The  increased  destruction  of  the  tissue-albumins  is  in 
such  cases  sufficiently  apparent  from  the  progressive  loss  of  flesh 
which  is  so  constantly  observed.  That  certain  nervous  influences 
may  here  be  at  work  is  probable,  and  we  know,  as  a  matter  of  fact, 
that  injury  to  a  certain  region  in  the  floor  of  the  fourth  ventricle 
is  invariably  followed  by  the  appearance  of  glucose  in  the  urine. 
But,  on  the  other  hand,  we  may  also  imagine  that  the  normal 
decomposition  of  the  sugar  is  prevented  owing  to  the  absence  of 
some  such  ferment  as  the  glucolytic  ferment  of  Lepine.  In  support 
of  this  view  is  the  fact  that  after  extirpation  of  the  pancreas  death 
invariably  results  with  symptoms  which  are  practically  identical 
with  those  seen  in  the  gravest  types  of  diabetes.  Ligation  of  the 
duct  does  not  produce  this  effect ;  and  it  is  noteworthy,  moreover, 
that  the  glucosuria  disappears  when  pieces  of  the  pancreas  are 
transplanted  under  the  skin  or  when  fresh  raw  pancreas  is  given 
with  the  food.  Within  the  past  ten  years  it  has  been  found  that  in 
a  not  inconsiderable  number  of  cases  of  diabetes  degenerative 
lesions  can  be  demonstrated  in  the  pancreas,  and  there  can  be  no 
doubt  at  the  present  time  that  a  certain  percentage  of  cases  are 
directly  referable  to  such  origin.  That  the  islands  of  Langerhans 
are  here  primarily  concerned  has  been  demonstrated  by  Opie.  In 
this  connection  Cohnheim's  researches  are  very  significant,  in  which 
he  could  show  that  while  neither  pancreas  nor  muscle-tissue  contain 
ferments  which  are  capable  of  splitting  glucose  by  themselves, 
extensive  glucolysis  is  effected  if  an  extract  of  pancreas  and  muscle- 
plasma  conjointly  are  allowed  to  act  upon  glucose.  A  condition 
thus  exists  which  seems  quite  analogous  to  the  relation  between 
trj'psin  and  enterokinase. 

In  the  milder  forms  of  diabetes  an  insufficiency  on  the  part  of  the 
muscle-tissue  manifestly  does  not  exist,  as  it  is  possible  to  prevent 
the  occurrence  of  glucosuria,  temporarily  at  least,  if  the  demand  for 
sugar  is  increased  by  abundant  muscular  exercise.  That  a  hepato- 
genic diabetes  may  coexist  with  a  myogenic  form,  cannot  be  doubted. 


THE  CARBOHYDRATES.  299 

This  is,  however,  not  the  place  to  enter  into  a  detailed  account  of 
the  mechanism  by  which  glucosuria  is  produced,  and  for  further 
information,  and  for  a  consideration  of  the  various  pathological  con- 
ditions under  which  sugar  may  be  found  in  the  urine,  the  reader  is 
referred  to  other  works. 

The  amount  of  sugar  which  may  be  present  in  the  urine  under 
pathological  conditions  is  exceedingly  variable.  On  the  one  hand, 
traces  only  may  be  found,  which  may  be  normal ;  while,  on  the  other 
hand,  the  daily  excretion  may  exceed  1000  grammes.  In  diabetes 
an  elimination  of  from  3  to  6  per  cent,  in  an  amount  of  urine 
varying  between  3000  and  6000  c.c.  may  be  regarded  as  moderate. 

Tests  for  Sugar. — Simple  tests  by  means  of  w4iich  glucose  can  be 
demonstrated  directly  in  the  urine  as  such  are,  unfortunately,  not 
available.  Other  sugars,  it  is  true,  enter  into  consideration  only 
under  exceptional  conditions,  but  if  it  is  desired  to  prove  that  the 
substance  which  gives  the  common  sugar  reactions  is  actually  glu- 
cose, a  more  detailed  examination  is  necessary.  Some  of  these  tests, 
moreover,  may  be  simulated  by  substances  which  are  not  carbo- 
hydrates, and  deductions  as  to  the  presence  or  absence  of  sugar  are 
hence  only  warrantable  when  these  can  be  excluded.  If  albumins 
are  present,  they  must  first  be  removed. 

Nylander's  Test. — This  test  is  to  be  preferred  to  the  more 
common  one  of  Trommer,  as  the  reagent  does  not  react  with  uric 
acid,  kreatiniii,  or  homogentisinic  acid.  With  many  of  the  conju- 
gate glucuronates,  however,  a  reduction  is  observed,  and  it  is  hence 
necessary  to  eliminate  this  source  of  error  when  a  positive  reac- 
tion is  obtained,  or  to  apply  additional  tests  in  which  this  possi- 
bility does  not  enter  into  consideration. 

The  reagent  is  prepared  as  follows  :  4  grammes  of  the  tartrate 
of  potassium  and  sodium,  together  with  2  grammes  of  subnitrate  of 
bismuth  and  10  grammes  of  sodium  hydrate  are  placed  in  90  c.c. 
of  water.  The  solution  is  heated  to  the  boiling-point,  filtered  on 
cooling,  and  is  then  ready  for  use.  It  is  kept  in  a  dark-colored 
bottle. 

A  few  cubic  centimeters  of  the  urine  are  treated  with  the  reagent, 
in  the  proportion  of  11:1,  and  boiled,  when  in  the  presence  of 
sugar  a  reduction  of  the  subnitrate  of  bismuth  to  bismuthous  oxide, 
or  even  to  the  metallic  form,  occurs.  As  a  consequence  the  mixture 
assumes  a  grayish,  dark-brown,  or  black  color,  and  on  standing  the 
precipitated  oxide  or  metal  settles  to  the  bottom  together  with  the 
earthy  phosphates. 

Fehling's  Test. — This  is  merely  a  modification  of  the  older 
Trommer's  test.  The  reagent  consists  of  two  solutions,  viz.,  one 
containing  34.64  grammes  of  copper  sulphate  in  500  c.c.  of  water, 
while  the  other  is  prepared  by  dissolving  173  granmies  of  tartrate 
of  potassium  and  sodium  and  125  grammes  of  caustic  soda  in  a  like 
amount  of  water.  Before  using,  equal  parts  of  the  two  solutions 
are  mixed  and  diluted  with  four  times  as  much  water.     A  few  cubic 


300  777^:   URISE. 

centimeters  of  the  resulting  reagent  are  boiled  and  treated  with  a 
small  amount  of  the  urine,  when  in  the  presence  of  sugar  yellow 
cuprous  hydroxide  or  red  cuprous  oxide  separates  out,  and  on  stand- 
ing settles  to  the  bottom.  After  the  addition  of  the  urine  the  solu- 
tions should  no  longer  be  boiled,  but  may  be  held  near  the  flame 
for  a  few  moments.  Unless  this  precaution  is  taken,  fallacious 
results  are  often  obtained,  as  uric  acid,  and  kreatinin  more  especially, 
may  cause  a  partial  reductif)n  of  the  copper  solution  on  prolonged 
boiling.  The  test  at  best  is  open  to  many  objections.  Conjugate 
glucuronates  and  homogentisinic  acid  likewise  give  a  positive  reac- 
tion, and  ammonia,  if  present  beyond  traces,  may  hold  in  solution 
any  cuprous  oxide  that  may  be  referable  to  very  small  amounts  of 
sugar. 

Fermentation  Test. — This  test,  when  controlled  by  Xylanders 
test,  is  the  most  satisfactory  one.  To  this  end,  a  little  compressed 
yeast  is  shaken  with  about  20  c.c.  of  urine,  and  the  mixture  is 
placed  in  a  saccharimetric  tube,  such  as  that  devised  by  Lohnstein 
or  Einhorn.  On  standing  at  the  ordinary  temperature  of  the  room, 
or,  still  better,  at  37°  C,  fermentation  occurs  if  glucose  is  present, 
and  the  liberated  carbon  dioxide  collects  at  the  top  of  the  tube. 
In  any  case,  however,  two  controls  should  be  made,  viz.,  one  to 
determine  that  the  yeast  is  active,  and  another  with  normal  urine. 
If  a  small  amount  of  sugar  is  present,  it  may  happen  that  the 
resulting  carbon  dioxide  is  absorbed.  If  in  such  a  case  Nylander's 
test  first  gave  a  positive  reaction,  but  no  longer  reacts  after  fermenta- 
tion is  complete  (in  twelve  to  twenty-four  hours),  the  presence  of 
sugar  may  V)e  inferred.  If,  on  the  other  hand,  no  fermentation 
occurs,  and  Xylander's  test  still  gives  a  positive  result,  we  may  con- 
clude that  the  reaction  is  due  to  the  presence  of  a  non-fermentable 
reducing  substance. 

Phenylhydrazin  Test. — As  has  been  pointed  out,  all  mono- 
saccharides and  some  of  the  disaccharides,  such  as  maltose,  isomal- 
tose,  and  lactose,  form  compounds  with  phenylhydrazin  which  are 
known  as  osazons  (see  page  (J7).  The  resulting  bodies  are  all  very 
similar,  but  may  be  distinguished  from  each  other  by  the  melting- 
point  of  their  crystals,  and  to  some  extent  also  by  their  microscopical 
appearance.  With  free  glucuronic  acid  a  similar  compound  may 
be  obtained,  according  to  Thierfelder,  wliich  may  also  be  recognized 
by  its  melting-point,  while  the  conjugate  glucuronates  are  inactive  in 
this  respect.  Pentoses  likewise  give  rise  to  the  formation  of  osa- 
zons, but  the  melting-point  of  the  resulting  crystals  serves  to  distin- 
guish these  also  from  the  osazons  of  the  hexases.  As  a  general 
rule,  however,  neither  the  pentoses  nor  glucuronic  acid  interferes 
with  the  reliability  of  the  test.  If  doubt  should  arise,  a  special 
examination  should  be  made  to  ascertain  w^iether  pentoses  or  glu- 
curonates are  present  in  amounts  sufficient  to  react  with  the  reagent. 
A  further  objection  to  the  phenylhydrazin  test  has  been  urged  on 


THE   CARBOHYDRATES.  301 

the  basis  that  its  delicacy  is  such  that  a  positive  reaction  is  obtained 
even  under  normal  conditions.     This,  however,  I  must  deny. 

The  test  is  conveniently  conducted  as  follows  °  5  drops  of  pure 
phenylhydrazin  are  mixed  in  a  test-tube  with  10  drops  of  glacial 
acetic  acid  and  I  c.c.  of  a  saturated  solution  of  common  salt.  To 
this  are  added  3  c.c.  of  urine,  when  the  mixture  is  boiled  for  two 
minutes  and  is  then  set  aside  to  cool.  In  the  presence  of  more 
than  0.5  per  cent,  of  glucose,  crystals  of  ])henyI-glu(?osazon  begin 
to  separate  out  after  one  or  two  minutes.  Should  smaller  amounts 
be  present,  it  is  necessary  to  wait.  The  sediment  is  then  exam- 
ined microscopically.  As  we  are  generally  only  dealing  with  glu- 
cose in  the  urine,  a  further  examination  is  usually  not  necessary, 
especially  if  the  substance  crystallizes  out  in  large  needles,  which  are 
often  collected  in  stars  and  sheaves.  To  identify  these  further,  how- 
ever, their  melting-point  must  be  determined.  As  has  been  stated, 
this  differs  in  the  different  osazons,  with  the  exception  of  laevulose 
and  glucose,  which  have  the  same  melting-point.  Lsevulose,  how- 
ever, is  found  only  under  exceptional  conditions.  Its  presence  may 
be  established  as  shown  below.  The  melting-points  of  the  various 
osazons  which  may  be  encountered  are  as  follows : 

Glucose 204°-205°  C. 

Lffivulose 204°-205°  C. 

Galactose 193°  C. 

Maltose 206°  C. 

Isomaltose      150°-153°  C. 

Lactose 200°  C. 

Arabinose 159°  C. 

Xvlose 159°  C. 

Giucuronicacid 114°-115°  C. 

The  glucosazon  is  insoluble  in  water,  but  dissolves  with  ease  in 
hot  alcohol,  from  which  it  can  be  precipitated  on  cooling  in  crystal- 
line form,  by  diluting  with  water.  The  crystals  are  then  collected 
on  a  filter,  dried  over  sulphuric  acid,  and  further  examined  if 
desired, 

PoLAEiMETRic  EXAMINATION. — The  polarimetric  examination 
for  the  presence  of  sugar  should  always  be  controlled  by  one  or 
more  of  the  tests  that  have  just  been  described.  Dextrorotation, 
unless  biliary  acids  are  present,  can  be  directly  referred  to  the 
presence  of  sugar,  and  usually  to  glucose.  Lsevorotation,  however, 
may  be  referable  to  other  reducing  substances  besides  Isevulose, 
such  as  the  conjugate  glucuronates,  /?-oxybutyric  acid,  and  others. 
If  such  substances,  moreover,  are  present  in  larger  amounts,  traces 
of  dextrose  may  be  overlookod.  It  is  hence  advisable  to  examine 
the  urine  both  before  and  after  treatment  w^ith  yeast,  and  in  doubt- 
ful cases  to  control  the  quantitative  results,  which  are  obtained  by 
the  polarimeter,  by  some  other  method.  For  a  detailed  description 
of  this  method  I  must  refer  the  reader  to  special  works.  In  every 
case  the  urine  must  be  perfectly  clear  and  free  from  albumin.     If 


302  THE   URINE. 

higlily  colored,  it  shoulfl  be  treated  with  lead  acetate  solution  and 
then  filtered,  in  which  case  allowance  must  be  made  for  the  degree 
of  dilution  if  quantitative  results  are  desired. 

Quantitative  Estimation. — Knapp's  Method. — This  method  is 
to  be  preferred  to  the  older  method  of  Fehling',  which  furnishes 
results  of  value  only  in  especially  experienced  hands. 

The  method  is  based  upon  the  observation  that  mercuric  cyanide 
in  alkaline  solution  is  reduced  by  sugar  to  metallic  mercury.  If 
urine  is  then  added  to  a  solution  containing  a  known  amount  of  the 
cyanide  until  this  is  entirely  reduced,  the  corresponding  amount  of 
sugar  can  be  directlv  ascertained. 

The  solution  which  is  generally  employed  for  this  purpose  con- 
tains 10  grammes  of  the  chemically  pure  cyanide,  and  100  c.c.  of  a 
solution  of  sodium  hydrate  (sp.  gr.  1.145)  in  the  liter:  20  c.c.  cor- 
respond to  0.05  gramme  of  glucose. 

The  urine  must  be  free  from  albumin  and  should  contain  not 
more  than  0.5  to  1  per  cent,  of  sugar.  This  should  first  be  ascer- 
tained by  a  preliminary  test.  If  more  is  present,  the  urine  should 
be  correspondingly  diluted. 

Twenty  c.c.  of  the  reagent  are  diluted  with  80  c.c.  of  distilled 
water,  or  with  less  if  a  smaller  amount  of  sugar  than  0.5  per  cent, 
is  present.  The  solution  is  heated  to  the  boiling-point,  and  then 
titrated  with  the  diluted  urine,  boiling  for  one-half  minute  after  the 
addition  of  every  2  c.c.  or  less  of  the  urine.  As  the  end-reaction 
is  approached,  the  mercury  together  with  the  phosphates  settles  to 
the  bottom  and  the  supernatant  fluid  becomes  clear.  The  final  point 
is  reached  when  a  drop  of  the  liquid,  placed  upon  filter-paper,  and 
successively  held  over  the  mouth  of  a  bottle  containing  fuming 
hydrochloric  acid  and  over  that  of  one  containing  a  strong  solution 
of  hydrogen  sulphide,  is  no  longer  colored  yellow.  The  results  are 
then  calculated  on  the  basis  outlined  above. 

Fehlixg's  Method. — Two  solutions  are  employed,  which  must 

be   kept  in  separate   bottles ;  the  one  contains  34.64  grammes  of 

crystallized  cupric  sulphate,  dissolved  in  500  c.c.  of  distilled  water, 

and  the  other  173  grammes  of  potassium  and  sodium  tartrate  and 

125  grammes  of  potassium  hydrate,  dissolved  in  an  equal  volume  of 

water.     This  solution  is  of  such  strength  that  tiie  copper  contained 

in  10  c.c.  (5  c.c.  of  each)  will  be  completely  reduced  by  0.05  gramme 

of  glucose.     If  then  urine  is  carefully  added  to  this  quantity  until 

complete  reduction  takes  place,  the  amount  of  sugar  contained  in  a 

given  specimen  of  urine  can  be  readily  calculated  according  to  the 

following  equation  : 

5 
y  :  0.05  :  :  100  :  x;     and  x  =  — , 

y 

in  which  y  indicates  the  number  of  cubic  centimeters  of  urine  re- 
quired to  reduce  the  10  c.c.  of  Fehling's  solution,  and  x  the  amount 
of  sugar  contained  in  100  c.c.  of  urine. 


THE  CARBOHYDRATES.  303 

As  the  best  results  are  obtained  only  if  from  5  to  10  c.c.  of  urine 
are  used  in  one  titration,  it  is  usually  necessary  to  dilute  the  urine 
to  the  required  degree  ;  in  the  determination  of  tiiis  point  the  specific 
gravity  may  serve  as  a  guide.  As  a  general  rule,  urines  of  a  specific 
gravity  of  1.030  should  be  diluted  five  times,  and  if  the  density  is 
still  higher  ten  times.  To  be  certain  that  the  proper  degree  of  dilu- 
tion has  been  reached,  5  c.c.  of  Fehling's  solution  are  treated  with 
1  c.c.  of  the  diluted  urine,  a  little  caustic  soda  solution  and  distilled 
water  beino-  added  to  make  in  all  about  25  c.c.  This  mixture  is 
thoroughly  boiled;  if  the  fluid  still  remains  blue,  another  1  c.c.  of 
diluted  urine  is  added,  and  so  on,  until  the  last  two  tests  difler  by 
1  c.c.  of  urine,  the  last  cubic  centimeter  added  causing  a  separation 
of  cuprous  oxide.  In  this  manner  the  percentage  of  sugar  may  be 
approximately  determined.  Albumin,  if  present,  must  first  be  re- 
moved by  boiling. 

10  c.c.  of  Fehling's  solution,  diluted  with  40  c.c.  of  water,  are 
placed  in  a  porcelain  dish  and  boiled.  While  boiling,  the  diluted 
urine  is  added  from  a  burette,  0.5  c.c.  at  a  time,  when,  as  a  rule, 
the  precipitated  cuprous  oxide  will  rapidly  settle,  so  that  gradually 
a  white  bottom  may  be  seen  through  the  blue  field,  the  color  of 
which  becomes  less  and  less  intense  upon  the  further  addition  of 
urine  until  finally  the  solution  is  almost  colorless.  When  this  point 
is  reached,  the  urine  is  added  drop  by  drop  until  decolorization 
is  complete.  The  degree  of  dilution  multiplied  by  5  and  the  result 
divided  by  the  number  of  cubic  centimeters  of  diluted  urine  em- 
ployed will  then  indicate  the  percentage-amount  of  sugar. 

Unfortunately,  it  is  at  times  difficult  to  determine  exactly  the  point 
when  all  the  copper  has  been  reduced — i.  c,  the  point  at  which  the 
blue  color  has  entirely  disappeared.  When  it  is  thought  that  this  has 
been  reached,  about  1  c.c.  should  be  filtered  through  thick  Swedish 
filter-paper,  and  the  filtrate  (which  must  be  absolutely  clear)  acidified 
with  acetic  acid  and  treated  with  a  drop  or  two  of  a  solution  of 
potassium  ferrocyanide.  If  unreduced  copper  is  still  present  in  the 
solution,  a  brown  color  will  result,  indicating  that  sufficient  urine 
has  not  been  added.  But  if,  on  the  other  hand,  no  brown  discolor- 
ation is  noted,  it  is  possible  that  the  desired  point  has  been  passed, 
when  the  titration  should  be  repeated. 

Differential  Density  Method. — The  specific  gravity  is  first 
determined  in  the  fresh  urine,  after  adding  2  grammes  of  tartrate 
of  potassium  and  sodium,  and  2  grammes  of  diacid  sodium  phos- 
phate to  every  100  c.c.  To  about  200  c.c.  which  have  thus  been 
prepared,  from  5  to  10  grammes  of  fresh  yeast  are  added,  and  the 
mixture  is  set  aside  at  a  tem})erature  of  from  20°  to  25°  C  until 
fermentation  is  com])leted.  If  but  little  sugar  is  present,  two  or 
three  hours  will  suffice  ;  otherwise  the  mixture  is  allowed  to  stand 
for  twelve  hours.  Evaporation  is  guarded  against  by  ck)sing  the 
bottle  with  a  perforated  stopper  through  which  a  finely  drawn  out 
tube  passes,  which  is  open  at  the  distal  end.     The  specific  gravity  is 


304  THE   URINE. 

then  airain  determined  at  the  same  time  as  before,  and  the  difference 
multiplied  by  0.230.  The  result  indicates  the  amount  of  sugar  in 
per  cent. 

The  method  yields  good  results  unless  very  small  amounts  of 
sugar  are  present,  viz.,  less  than  O.o  per  cent.  In  such  an  event, 
the  reducing  power  of  the  urine  is  first  ascertained  according  to 
Fehling's  or  Knapp's  method.  It  is  then  fermented,  when  the 
remaining  reducing  substances  are  again  determined.  The  differ- 
ence may  be  referred  to  sugar. 

Fermentation  Method. — In  the  clinical  laboratory  especially 
constructed  saccharimetric  tubes  are  used,  of  which  Lohnstein's  is 
probably  the  best.  These  are  provided  with  a  scale  which  enables 
the  percentage  of  sugar  to  be  read  off  directly  from  the  amount  of 
carbonic  acid  that  has  gathered  in  the  upper  end  of  the  tube.  The 
instruments  are  accompanied  by  printed  instructions,  which  need 
not  be  considered  at  this  place. 

Lactose. — The  presence  of  lactose  in  the  urine  is  a  normal 
occurrence  in  nursing  women,  and  it  is  at  times  found  also  imme- 
diately preceding  confinement.  Its  appearance  in  the  urine  is  un- 
doubtedly referable  to  absorption,  owing  to  the  fact  that  a  super- 
abundance of  milk  is  being  produced,  and  we  accordingly  also  find 
the  substance  in  the  urine  when  for  any  reason  lactation  is  sup- 
pressed. Once  it  has  found  its  way  into  the  circulation,  its  elimina- 
tion through  the  kidneys  necessarily  follows,  as  the  body  is  incapable 
of  inverting  the  disaccharides  to  monosaccharides. 

Aside  from  its  occurrence  in  connection  with  lactation,  lactose 
is  found  in  the  urine  only  if  abnormally  large  amounts  have  been 
ingested.  In  such  an  event,  as  has  been  stated,  a  certain  proportion 
of  the  sugar  escapes  inversion  in  the  epithelial  cells  which  line  the 
intestinal  tract,  and  on  entering  the  general  circulation  it  is  elimi- 
nated as  such.  The  amount  of  lactose  which  may  be  found  in  the 
urine  of  nursing  women  varies  between  0.013  and  0.438  per  cent. 
Its  presence  in  the  urine  may  be  suspected  if  the  reduction  test  and 
the  phenylhydrazin  test  yield  a  positive  result,  while  the  fermenta- 
tion test,  as  usually  conducted,  is  negative.  Like  glucose,  the  sub- 
stance is  dextrorotatory.  To  identify  the  sugar  positively  as  lactose, 
however,  it  is  necessary  to  isolate  it  as  such. 

Isolation. — The  collected  urine  of  twenty-four  hours  is  precipi- 
tated with  lead  subacetate  and  filtered.  After  washing  with  water 
the  filtrate  and  washings  are  mixed  and  treated  with  ammonia. 
The  resulting  precipitate  is  filtered  off  and  the  filtrate  again  pre- 
cipitated with  lead  subacetate  and  ammonia,  and  so  on  until  the 
final  filtrate  is  optically  inactive.  The  precipitates,  with  the  excep- 
tion of  the  first,  are  then  mixed,  washed  with  w^ater,  decomposed 
with  hydrogen  sulphide,  and  filtered.  In  the  filtrate  the  excess  of 
hydrogen  sulphide  is  removed  by  a  current  of  air,  and  freed  from 
any  acids  that  have  been   liberated  by  shaking  with  argentic  oxide. 


THE  CARBOHYDRATES.  305 

The  mixture  is  filtered,  freed  from  soluble  silver  with  hydrogen  sul- 
phide, treated  with  barium  carbonate,  and  concentrated  to  a  small 
volume ;  90  per  cent,  alcohol  is  then  added,  which  causes  the 
formation  of  a  flocculent  precipitate.  This  is  filtered  oif.  The 
filtrate  is  placed  in  the  desiccator,  when  on  standing  crystals  of 
lactose  gradually  separate  out.  These  may  be  purified  by  recrys- 
tallization,  decolorization  with  animal  charcoal,  and  extraction  with 
60-70  per  cent,  alcohol. 

Lsevulose. — The  occurrence  of  a  Isevorotatory  sugar  has  been  at 
times,  though  rarely,  observed  in  the  urine  of  diabetic  patients, 
where  it  was  present  either  alone  or  in  association  with  glucose. 
Like  dextrose,  the  substance  reduced  Fehling's  and  Nylander's  solu- 
tion, and  formed  an  osazon  with  phenylliydrazin,  with  a  melting- 
point  of  205°  C  It  was  fermentable,  but,  unlike  true  lsevulose, 
could  be  precipitated  with  basic  lead  acetate. 

Leo's  laiose,  which  was  also  obtained  from  a  diabetic  urine  on 
one  occasion,  was  very  similar  to  the  body  just  described,  but,  unlike 
this,  could  not  be  fermented.  AVith  phenylliydrazin,  moreover,  it 
formed  a  yellowish-brown  non-crystallizable  oil. 

Of  the  nature  of  these  bodies  nothing  further  is  known. 

The  presence  of  a  Isevorotatory  sugar  can,  of  course,  readily  be 
established  by  the  common  tests,  supplemented  by  a  polarimetric 
examination,  if  it  is  present  alone.  If  glucose,  however,  also  is  con- 
tained in  the  urine  in  amounts  sufficient  to  counteract  the  Isevorota- 
tion,  the  matter  is  more  difficult.  In  such  an  event,  however,  it  wdll 
be  observed  that  higher  values  are  obtained  in  estimating  the  sugar 
with  Knapp's  method,  or  according  to  the  diJBPerential  density  method, 
than  with  the  polarimeter,  for  reasons  which  are  self-evident. 

According  to  Neuberg  and  Strauss,  the  presence  of  lsevulose  can 
be  definitely  established  in  the  urine,  the  blood-serum,  ascitic  fluid, 
or  in  pleural  eifusions  by  its  transformation  into  the  cofresponding 
methyl-phenyl-osazon,  which  can  be«obtained  in  crystalline  form 
(see  page  69).^ 

Lsevulose  also  gives  Seliwanoffs  reaction,  viz.,  it  gives  rise  to  the 
formation  of  a  red  pigment  on  boiling  with  resorcin  and  hydro- 
chloric acid,  which  is  soluble  in  alcohol.  The  reaction  in  question 
is  common  to  all  the  ketoses  of  the  6 — carbohydrate  series  (fructose 
and  sorbinose),  but  is  also  obtained  with  those  polysaccharides  which 
yield  fructose  on  hydrolysis,  viz.,  cane-sugar,  raffinose,  inulin,  etc. 

Maltose. — Maltose  together  with  glucose  was  found  on  one  occa- 
sion in  the  urine  of  a  patient  supposedly  the  subject  of  pancreatic 
disease.  Its  recognition  is  essentially  dependent  upon  the  forma- 
tion of  its  osazon,  and  the  identification  of  the  latter  by  its  melting- 
point  (see  also  page  72). 

Dextrin. — That  traces  of  dextrin  are  found  in  the  urine  under 
normal  conditions  has  been  pointed  out.  Larger  amounts  have  been 
observed  in  the  case  of  a  diabetic  patient,  where  the  substance 
apparently  took  the  place  of  glucose.     Of  its  origin  nothing  definite 

»  The  method  is  described  in  my  Clinical  Diagnosis,  oth  ed. 
20 


306  THE   URINE. 

is  known,  but  it  is  likely  that  in  health  the  substance  gains  entrance 
to  the  circulation  in  a  more  or  less  accidental  way,  and  is  then, 
of  course,  eliminated  at  once.  It  is  now  regarded  as  identical  with 
the  animal  gum  of  Landwehr. 

To  demonstrate  the  presence  in  normal  urine  of  carbohydrates  of 
this  character,  the  urine  is  boiled  with  dilute  sulphuric  acid  for  about 
thirty  minutes,  and  after  being  rendered  alkaline  with  sodium  hydrate 
is  examined  with  Xylander's  test.  A  positive  reaction  may  now  be 
obtained,  while  previously  no  reduction  occurred. 

For  the  isolation  of  these  normal  carbohydrates  the  reader  is 
referred  to  special  works. 

Pentoses. — That  traces  of  pentoses  may  occur  in  the  urine  under 
normal  conditions  has  been  stated.  As  has  been  shown,  the 
pancreas  contains  a  nucleoproteid  which  yields  a  very  large 
amount  of  pentose  on  decomposition,  and  one  might  imagine 
that  the  normal  pentose  of  the  urine  might  possibly  be  refer- 
able to  this  source.  Neuberg,  however,  has  shown  that  the 
two  are  not  identical.  The  urine  pentose  (arabinose)  is  optically 
inactive,  while  the  pentose  of  the  pancreas  is  ^-xylose.  The  normal 
pentoses,  no  doubt,  are  referable  to  the  ingestion  of  such  articles  of 
food  as  prunes,  cherries,  grapes,  beer,  wine,  etc.  As  in  the  case  of 
glucose,  the  power  to  assimilate  pentoses  seems  to  vary  with  differ- 
ent individuals,  and  here,  as  there,  a  digestive  pentosuria  can  be 
artificially  produced.  In  diabetes  the  power  to  oxidize  the  pentoses 
may  be  much  impaired,  and,  very  curiously,  the  largest  quantities 
have  been  observed  in  morphin  habitues. 

The  individual  pentoses  which  have  thus  far  been  encountered  in 
the  urine  are  arabinose,  xylose,  and  rhamnose.  They  all  reduce 
Fehling's  solution,  and  give  rise  to  osazons  with  phenylhydrazin. 
The  melting-points  of  the  resulting  compounds,  however,  are  differ- 
ent from  those  of  the  common  hexosazons  (see  page  301).  In  the 
amounts,  however,  in  which  they  are  usually  present  no  reactions 
are  obtained  in  this  manner.  The  fermentation  test  is  not  obtained. 
Xylose  and  rhamnose  turn  the  plane  of  polarization  to  the  right, 
while  arabinose  is  optically  inactive. 

Of  special  interest  is  the  fact  that  as  a  result  of  a  fermenta- 
tive splitting  off  of  CO2  glucuronic  acid  passes  over  into  the  aldo- 
pentose  /-xylose ;  in  this  manner  the  possible  transformation  of 
a  suo-ar  of  the  c?-series  into  one  of  the  ^-series  has  been  demon- 
st  rated. 

To  demonstrate  the  presence  of  pentoses  in  the  urine  the  follow- 
ing test  of  Tollens  is  employed  : 

Orcin  Test  (as  described  by  Bial). — 4-5  c.c.  of  the  reagent 
(500  c.c.  of  30  per  cent,  hydrochloric  acid,  1  gramme  of  orcin,  and 
25  drops  of  liquor  ferri  sesquichloridi)  are  heated  to  boiling ;  the  tube 
is  removed  from  the  flame  and  a  few  drops  to  1  c.c.  of  .urine  added. 
With  ])entose  urine  there  is  immediately  a  fine  green  coloration. 
Normal  and  diabetic  urines  do  not  give  the  reaction,  nor  do  urines 


THE  ALBUMINS.  307 

containing  conjugate  glucnronates.     The  reagent  keeps  for  at  least  a 
year.^ 

With  Tollens'  phloroghicin  test,  which  is  conducted  in  the  same 
manner,  a  deep-red  color  develops  instead ;  but  this  reaction  is  also 
common  to  the  glucnronates.  (The  reagent  is  prepared  in  the  same 
manner  as  in  the  case  of  the  orcin  reagent,  phloroglucin  being  simply- 
substituted  for  the  orcin.) 

THE  ALBUMINS. 

As  every  urine  contains  a  small  number  of  cellular  elements 
which  are  derived  from  the  urinary  tract,  it  can  readily  be  under- 
stood that  even  under  normal  conditions  albumin  can  be  demon- 
strated with  suitable  methods.  The  amount,  however,  is  exceed- 
ingly small,  and  with  the  common  tests  a  jjositive  reaction  cannot 
be  obtained  unless  the  substance  in  question  has  been  previously 
isolated  from  a  large  quantity  of  urine.  In  such  an  event  a  trace 
of  a  nucleo-albumin  can  be  demonstrated.  The  occurrence  of  the 
common  albumins  of  the  blood,  on  the  other  hand,  is  always  a 
pathological  phenomenon.  Some  writers,  it  is  true,  speak  of  a 
physiological  albuminuria,  which  may  be  observed  after  severe 
muscular  exercise,  following  cold  baths,  during  pregnancy,  etc., 
and  it  is  even  claimed  that  the  elimination  of  albumin  in  young 
persons,  which  is  so  commonly  observed  in  neurotic  and  anaemic 
individuals,  in  association  with  an  increased  amount  of  uric  acid 
and  oxalic  acid,  belongs  to  this  order.  There  is  an  increasing 
tendency  among  pathologists,  however,  to  doubt  the  physiological 
character  of  such  forms  of  albuminuria,  and  personally  I  main- 
tain that  every  albuminuria  of  a  hsematogenic  type  is  a  pathological 
phenomenon.  We  may,  in  fact,  go  further,  and  assume  that  the 
appearance  of  nucleo-albumin  also  in  amounts  which  can  be  de- 
monstrated by  ordinary  tests  is  abnormal,  as  such  an  occurrence 
must  of  necessity  be  associated  with  an  increased  desquamation  of 
epithelial  cells,  which  in  itself  is  evidence  of  a  pathological  process. 
This,  however,  is  hardly  the  place  to  enter  upon  a  detailed  con- 
sideration of  the  various  morbid  conditions  in  which  albuminuria 
may  occur,  and  it  will  suffice  to  state  that  any  disturbance  in  the 
nutrition  of  the  glandular  elements  of  the  kidney,  from  whatever 
cause,  will  at  once  find  expression  in  the  appearance  of  albumin  in 
the  urine.  The  albumins  which  are  then  eliminated  are  the  common 
albumins  of  the  blood,  and  notably  serum-albumin  and  serum- 
globulin.  Fibrinogen,  on  the  other  hand,  is  usually  not  found.  In 
cases  of  hgeraaturia  and  chyluria,  however,  its  presence  may  be 
inferred  from  the  formation  of  coagula  of  fibrin.  This  may  occur 
in  the  urinary  passages  already,  but  more  commonly  it  is  observed 
after  the   urine  has  been  voided. 

Other  albumins  besides  those  which  are   normally  found  in  the 
blood  are  encountered  only  exceptionally   in   the  urine ;  but  it  may 


308  THE   URINE. 

be  stated  that  whenever  such  substances  find  their  way  into  the 
general  circulation  their  elimination  at  once  follows.  Formerly 
it  was  taught  that  peptones  could  thus  appear,  and  for  many  years 
various  types  of  peptonuria  were  described.  More  recent  investiga- 
tions, however,  have  shi:)wn  that  the  substances  in  question  were  in 
reality  no  peptones  in  the  sense  of  Kiihne,  but  albumoses.  Some 
of  these  are,  no  doubt,  identical  with  the  common  digestive  albu- 
moses, and  find  their  way  into  the  blood,  when  their  further  trans- 
formation into  native  albumins  does  not  occur  in  the  epithelial 
cells  of  the  digestive  tract.  Others  again  are  probably  formed  in 
the  body  proper  in  diseases  which  are  associated  with  suppurative 
processes,  and  in  which  the  formation  of  albumoses  occurs  at  the 
expense  of  the  tissue  albumins  under  the  influence  of  various  micro- 
organisms. Under  still  other  conditions,  as  in  the  various  non- 
septic  fevers,  in  phosphorus  poisoning,  etc.,  the  albumosuria  may 
be  the  expression  of  a  metabolic  abnormality  per  se,  and  is  possibly 
dependent  upon  the  action  of  the  various  tissue  ferments. 

Of  special  interest,  further,  is  the  appearance  in  the  urine  of  the 
so-called  albumin  of  Bence  Jones,  which  has  been  repeatedly  ob- 
served in  association  with  multiple  myelomata  of  the  bones.  Of  its 
chemical  nature,  however,  little  is  known.  By  some  it  is  regarded 
as  an  albumose,  but,  according  to  Neumeister,  it  is  not  identical 
with  any  of  the  known  digestive  albumoses.  I  shall  revert  to  it 
later. 

In  diseases,  finally,  in  which  an  increased  destruction  of  leuco- 
cytes is  taking  place,  both  histon  and  nucleohiston  have  been  found. 

Of  other  albumins  which  are  foreign  to  the  blood,  only  egg- 
albumin  has  been  encountered,  following  the  ingestion  of  excessive 
amounts  of  the  substance. 

Tests  for  the  Common  Albumins  of  the  Blood. — The  Nitric 
Acid  Test. — A  small  amount  of  urine  is  placed  in  a  conical  glass 
and  is  underlaid  with  a  few  cubic  centimeters  of  concentrated  nitric 
acid,  when  in  the  presence  of  serum-albumin  and  serum-globulin  a 
white,  opaque  disk  of  coagulated  albumin  is  formed  at  the  zone  of  con- 
tact, which  varies  in  intensity  and  extent  with  the  amount  of  albumin 
present.  Immediately  below  this  variously  colored  rings  also  are 
observed,  which  are  in  part  referable  to  the  decomposition  of 
indoxyl  and  skatoxyl  sulphate,  and  the  oxidation  of  the  liberated 
indoxyl  and  skatoxyl  to  blue  and  red  pigments.  In  the  presence 
of  bile-pigment  a  green  color  will  then  also  be  noted.  If  much 
urea  be  present  at  the  same  time,  it  may  happen  that  after  a  few 
minutes  a  dense  disk  of  urea  nitrate  crystals  separates  out  in  the 
lower  pigmented  layer,  but  more  conmionly  these  are  formed 
throughout  the  mixture  on  standing,  and  gradually  settle  to  the 
bottom.  Should  uric  acid,  further,  be  present  in  increased  amount, 
a  white  disk  develops  higher  up  in  the  urine,  and  separated  from 
that  referable  to  albumins  by  a  layer  of  clear  urine.  This  may  at 
times  be  quite  marked,  and  may  extend  downward  toward  the  nitric 


THE  ALBUMINS.  309 

acid  so  rapidly  that  it  is  difficult  to  say  whether  it  is  referable  to 
albumin  or  a  larg;e  excess  of  uric  acid.  Should  this  occur,  it  is  best 
to  dilute  the  urine  with  an  equal  volume  of  water,  or,  even  more 
stronglv,  when  a  portion  of  the  uric  acid  at  least  is  prevented  from 
separating  out  or  is  held  in  solution  altogether. 

As  nucleo-albumins,  when  present  beyond  traces,  can  simulate 
the  true  albumin  reaction,  it  is  Avell  to  dilute  the  urine  with  water 
and  to  examine  again  when  its  presence  is  suspected.  If  then  the 
reaction  is  more  pronounced  than  before,  the  precipitate  may,  in  part 
at  least,  be  referable  to  this  source.  This  possibility  should  be  con- 
sidered if  the  urine  contains  an  increased  number  of  morphological 
elements,  and  if  the  reaction  is  slight.  Other  tests  should  then  also 
be  employed. 

All)umoses,  if  present  beyond  traces,  also  react  with  nitric  acid, 
but  it  is  to  be  noted  that  in  such  cases  the  precipitate  disappears  on 
heating  and  reappears  on  cooling,  while  the  liquid  at  the  same  time 
assumes  an  inteusely  yellow  color.  Should  a  mixed  albuminuria 
exist — i.  €.,  should  albumoses  and  albumin  be  present  simultane- 
ously— the  clearing  of  the  urine  is  only  partial. 

As  nitric  acid  also  precipitates  certain  resins  which  may  have 
been  administered  for  medicinal  purposes,  it  is  at  times  necessary  to 
eliminate  this  possibility  of  error.  Their  presence  is  indicated  if 
the  precipitate  disappears  on  shaking  the  mixture  with  ether. 

The  Boiling  Test. — The  urine  should  present  a  Teebly  acid  or 
neutral  reaction.  If  alkaline,  it  is  rendered  nearly  neutral  by  adding 
a  drop  or  two  of  dilute  acetic  acid.  A  few  cubic  centimeters  are 
then  boiled  in  a  test-tube,  when  in  the  presence  of  coagulable 
albumins  the  liquid  becomes  turbid,  and  on  standing  a  flocculent 
precipitate  gathers  at  the  bottom  of  the  tube.  The  turbidity  may, 
however,  at  times  be  due  to  a  precipitation  of  neutral  earthy  phos- 
phates. To  distinguish  between  the  two,  one  or  two  drops  of  a  25 
per  cent,  solution  of  nitric  acid  are  now  added  for  every  1  c.c.  of 
the  urine.  The  earthy  phosphates  are  thus  dissolved,  while  the  pre- 
cipitate of  albumin  remains  unaffected.  If  more  than  traces  of 
albumin  are  present,  this  test  is  very  reliable  ;  otherwise  there  is 
danger  of  dissolving  the  small  amount  of  albumin.  If  nitric  acid 
is  used  instead  of  acetic  acid,  this  danger  is  generally  small,  how- 
ever, but  the  possibility  exists,  nevertheless.  Hence  in  doubtful 
cases  it  is  always  best  to  resort  to  the  nitric  acid  test  as  well. 

If  the  albumin  of  Bence  Jones  should  be  present,  coagulation 
occurs  at  a  temperature  of  50°  C,  already,  but  it  will  be  noted 
that  the  precipitate  disappears  on  subsequent  boiling  and  reappears 
on  cooling. 

The  common  albumoses,  as  well  as  nucleo-albumin,  are  not 
thrown  down.  The  presence  of  the  former  may  be  inferred  if 
after  the  addition  of  the  acid  and  subsequent  cooling  a  white  pre- 
cipitate is  formed,  which  dissolves  upon  the  application  of  heat  and 
reappears  on  cooling.    * 


310  THE  URINE. 

If  acetic  acid  is  to  be  employed  instead  of  nitric  acid,  it  is  best 
to  treat  the  urine  with  one-sixth  of  its  vohime  of  a  saturated  solu- 
tion of  common  salt,  after  having  rendered  it  distinctly  acid.  It 
is  then  boiled  as  before.  In  this  case  the  danger  of  dissolving  the 
precipitated  albumins  is  much  lessened. 

The  Potassium  Ferrocyanide  Test. — A  few  cubic  centimeters  of 
urine  are  strongly  acidified  with  acetic  acid,  and  treated  with  a 
10  per  cent,  solution  of  potassium  ferrocyanide  drop  by  drop,  when 
in  the  presence  of  albumin  a  precipitation  occurs  which  varies 
in  intensity  with  the  amount  present.  Concentrated  urines  should 
first  be  diluted.  Albumoses  are  also  thrown  down,  but  are  redis- 
solved  on  boiling  and  reappear  on  cooling.  Should  nucleo-albu- 
mins  be  present  beyond  traces,  a  precipitate  develops  upon  the 
addition  of  the  acetic  acid.  This  m'ay  also  occur  if  urates  are 
present  in  large  amounts.  But  in  this  event  the  precipitate  clears 
upon  warming  the  solution  ;  and  if  the  urine,  moreover,  is  previously 
diluted,  it  does  not  occur  at  all,  while  the  separation  of  nucleo-albu- 
min  takes  place  more  rapidly  in  the  latter  case  than  before. 

Still  other  tests  exist  which  are  equally  good,  but  for  practical 
purposes  the  three  just  described  will  suffice.  In  every  case, 
however,  it  is  necessary  that  the  urine  should  be  perfectly  clear. 
If  turbid,  owing  to  precipitation  of  any  of  the  normal  constit- 
uents of  the  urine,  simple  filtration  generally  suffices  ;  but  if  refer- 
able to  bacteria,  it  is  best  shaken  with  powdered  talcum  and  then 
filtered. 

Special  Test  for  Serum- albumin. — If  it  is  desired  to  demonsti'ate 
the  presence  of  serum-albumin  by  itself,  the  urine  is  rendered 
amphoteric  or  faintly  alkaline  with  sodium  hydrate,  and  satu- 
rated with  magnesium  sulphate  to  remove  any  globulins  that  may 
be  present.  The  filtrate  is  then  acidified  with  acetic  acid  and  boiled, 
when  in  the  presence  of  serum-albumin  precipitation  occurs. 

Special  Test  for  Serum-globuliti. — This  is  conducted  as  above, 
or  by  adding  an  equal  volume  of  a  saturated  solution  of  ammo- 
nium sulphate  to  the  amphoteric  urine,  when  the  globulins  are 
thrown  down.  Urates  may  then  also  separate  out,  but  this  always 
occurs  later.  The  precipitated  globulins  are  soluble  in  acetic  acid. 
As  I  have  stated  before,  there  is  one  instance  of  globulinuria  on 
record  in  Mhich  the  substance  was  found  in  the  sediment  in  crystal- 
line form. 

Test  for  Nucleo-albumin. — A  small  amount  of  urine  is  diluted 
with  water  and  then  treated  drop  by  drop  with  strong  acetic  acid. 
In  this  manner  the  precipitation  of  urates  is  prevented,  while  the 
nucleo-albumin  separates  out.  To  identify  it  as  such,  however,  it  is 
necessary  to  isolate  the  substance  in  larger  amounts.  To  this  end, 
the  collected  urine  of  tAventy-four  hours  is  carefully  neutralized  and 
concentrated  to  about  1000  c.c.  at  a  temperature  of  60°-70°  C.  On 
filtering,  it  is  saturated  with  ammonium  sulphate  in  substance  and 
the  precipitate   collected  on  a   filter.     This  is  dissolved  in  a  little 


THE  ALBUMINS.  311 

water  and  freed  from  salts  by  dialysis.  Should  hetero-albumose  be 
present,  this  separates  out  and  is  removed  by  filtration.  A  portion 
of  the  remaininsx  solution  is  then  tested  with  acetic  acid,  as  described  ; 
the  precipitate  should  be  soluble  in  mineral  acids.  In  the  remaining 
solution  the  body  is  completely  thrown  down  with  acetic  acid.  The 
precipitate  is  filtered  off,  washed  with  dilute  acetic  acid,  and  dried. 
On  fusion  with  caustic  alkali  and  potassium  nitrate,  phosphoric  acid 
should  then  be  liberated  if  the  substance  is  a  nucleo-albumin.  If 
this  reaction  is  not  obtained,  but  if  on  boiling  with  dilute  mineral 
acids  a  reducing  substance  is  set  free,  it  may  be  assumed  that  the 
body  in  question  is  mucin. 

Test  for  Albumoses. — To  test  for  albumoses  in  general,  a  small 
amount  of  the  urine  is  acidified  with  acetic  acid  and  treated  with  an 
equal  volume  of  a  saturated  solution  of  common  salt.  The  solution 
is  then  boiled  and  filtered  while  still  hot,  so  as  to  remove  any  coagu- 
lable  albumins  that  may  be  present.  On  cooling,  the  albumoses 
separate  out,  but  redissolve  on  boiling.  In  such  an  event,  the  solu- 
tion also  gives  the  biuret  reaction  and  that  of  ]\Iillon. 

Safer,  however,  is  the  following  method,  which  should  always  be 
employed  when  there  is  reason  to  believe  that  albumoses  are  present 
only  in  traces.  The  collected  twenty-four  hours'  urine  is  carefully 
neutralized,  concentrated  to  about  1000  c.c.  at  60°-70°  C,  filtered, 
and  saturated  with  ammonium  sulphate  in  substance.  The  pre- 
cipitate is  collected  on  a  filter,  and  dissolved  in  a  little  water,  when 
a  small  portion  is  treated  with  an  equal  volume  of  a  saturated  solu- 
tion of  common  salt,  and  with  acetic  acid  or  nitric  acid  drop  by 
drop  so  long  as  any  precipitate  that  has  formed  is  thus  increased. 
The  solution  is  then  boiled.  If  coagulable  albumins  are  present, 
these  are  precipitated,  and  are  filtered  otf  from  the  hot  solution.  If 
the  filtrate  becomes  turbid  again  on  cooling,  and  clears  upon  sub- 
sequent boiling,  the  presence  of  albumoses  may  be  inferred.  To 
determine  the  character  of  the  albumoses  in  question,  the  remaining 
liquid  is  dialyzed  (see  above),  freed  from  nucleo-albumin  by  means 
of  acetic  acid,  neutralized,  concentrated  on  a  water-bath,  and 
saturated  with  rock-salt.  If  primary  albumoses  are  present,  they 
are  thus  precipitated  and  filtered  off.  When  acetic  acid  that  has 
been  saturated  with  common  salt  is  added  to  the  filtrate  the  deutero- 
albumoses  are  thrown  down.  This  test  should  be  applied  in  the 
preliminary  examination  also  if  no  reaction  is  obtained. 

Instead  of  using  sodium  chloride  to  precipitate  the  albumoses, 
ammonium  sulphate  can,  of  course,  also  be  used,  as  has  been 
described  on  page  198. 

True  peptones,  in  the  sense  of  Kiihne,  do  not  occur  in  the 
urine,  and  it  is  hence  unnecessary  to  describe  the  older  and  more 
complicated  methods  which  formerly  were  employed  in  their  search. 

Bence  Jones'  Albumin. — This  body,  as  has  been  pointed  out,  has 
repeatedly  been  encountered  in  the  urine  in  association  with  the 
'existence  of   multiple    myelomata    of   the    bones.      Of  its    nature, 


312  THE   URINE. 

however,  little  is  known.  Most  observers  have  regarded  it  as  an 
albuniose,  but  it  is  admitted  that  it  is  not  identical  witli  any  of  the 
known  digestive  alburaoses.  Like  the  globulin  described  by  Paton, 
it  lias  been  found  in  crystalline  form  in  the  urinary  sediment,  and 
can  be  made  to  crystallize  after  its  isolation  in  amorphous  form. 
Magnus-Levy,  who  has  recently  studied  the  body  in  question,  while 
likewise  unable  to  identify  it  with  any  of  the  known  albumins  or 
albumoses,  points  out  that  it  has  in  realitv  only  one  property  in 
common  with  the  alburaoses,  viz.,  the  solubility  of  its  precipitate  on 
boiling.  He  points  out,  however,  that  this  is  only  apparent,  and 
that  under  suitable  conditions  the  body  is  coagulated  on  heating  to 
100'^  C,  like  the  native  albumins.  He  further  noted  that,  like  the 
true  albumins,  the  substance  yields  the  common  digestive  products 
of  these  bodies,  viz.,  primary  and  secondary  albumoses  ;  but,  as  in 
the  case  of  casein,  no  hetero-group  could  be  demonstrated.  Those 
results  I  have  personally  confirmed,  and  it  is  thus  conclnsivoly 
established  that  the  body  cannot  be  an  albumose.  Pending  further 
investigations,  it  is  hence  advisable  to  term  the  substance  the 
albumin  of  Bence  Jones.  Of  its  origin  nothing  definite  is  known. 
The  amount  which  is  often  found,  however,  is  so  large  that  the  con- 
clusion suggests  itself  that  the  substance  may  be  derived  from  the 
ingested  albumins,  and  is  formed  in  the  intestinal  canal  or  its 
walls  as  a  result  of  some  abnormal  digestive  process.  The  pres- 
ence of  the  albumin  may  be  suspected  if  a  urine  gives  the  usual 
albumose  reaction  to  a  marked  degree,  as  the  disease  in  question  is 
in  reality  the  only  one  in  which  larger  amounts  of  an  ''  albumose"- 
like  body  are  obtained.  It  can  then  be  isolated  by  treating  the 
neutralized  urine  with  double  its  volume  of  a  saturated  solution  of 
ammonium  sulphate.  To  identify  the  substance,  it  is  advisable  to 
digest  the  body  with  pepsin,  to  demonstrate  the  formation  of  proto- 
albumose,  and  to  show  that  no  hetero-albumose  is  produced. 

Test  for  Fibrin. — When  fibrin  is  present  in  the  urine,  it  usually 
occurs  iu  the  form  of  distinct  clots,  the  nature  of  which  is  commonly 
apparent  without  chemical  examination.  If  it  is  to  be  identified  in 
this  manner,  however,  the  clots  are  washed  with  water  until  free 
from  blood-pigments.  They  are  then  placed  in  a  5  per  cent,  solu- 
tion of  sodium  chloride  containing  an  excess  of  thymol,  to  guard 
against  putrefactive  changes.  It  will  be  observed  that  the  substance 
does  not  dissolve,  while  in  a  0.3  per  cent,  solution  of  hydrochloric 
acid  it  rapidly  swells  and  is  digested  after  the  addition  of  a  little 
pepsin. 

Test  for  Histon. — The  twenty-four  hours'  urine  is  first  freed 
from  coagulable  albumins  by  boiling.  It  is  then  precipitated  with 
a  large  excess  of  94  per  cent,  alcohol.  The  precipitate  is  washed 
with  hot  alcohol  and  dissolved  in  boiling  water.  On  cooling,  the 
solution  is  acidified  with  hydrochloric  acid  and  allowed  to  stand  for 
a  number  of  hours.  Any  uric  acid  that  has  separated  out  is 
removed  by  filtration,  when  the  filtrate  is  precipitated  with  ammonia.  * 


THE  PIGMENTS  OF  THE   URINE.  313 

The  collected  material  is  washed  with  ammoniacal  water  until 
the  washings  no  lonsjer  give  the  biuret  reaction.  It  is  then  dissolved 
in  dilute  acetic  acid.  If  histon  is  present,  the  solution  coagulates  on 
boiling  and  gives  the  biuret  reaction.  The  coagulated  material  dis- 
solves in  mineral  acids. 

Quantitative  Estimation  of  the  Coagulable  Albumins. — In 
the  clinical  laboratory  the  so-called  album  in  imeters  of  Esbach  are 
conveniently  employed  for  this  purpose.  The  method  is  exceedingly 
simple,  and  gives  results  which  are  sufficiently  accurate  for  ordinary 
purposes.  To  this  end,  the  tube  is  filled  with  urine  to  the  mark  U. 
Esbach's  reagent,  which  consists  of  an  aqueous  solution  containing 
10  grammes  of  picric  acid  and  20  grammes  of  citric  acid  to  the 
liter,  is  then  added  to  the  mark  R.  The  tube  is  closed,  inverted  a 
number  of  times,  and  set  aside  for  twenty-four  hours.  The  number 
of  the  scale  which  corresponds  to  the  height  of  the  precipitated 
albumins  indicates  the  amount  in  grammes  in  1000  c.c.  of  urine. 
Care  must  be  had,  however,  that  the  urine  is  acid,  that  the  density 
does  not  exceed  1.006-1.008,  and  that  the  temperature  remains  at 
about  15°  C. 

If  more  accurate  results  are  desired,  a  known  volume  of  urine, 
feebly  acidified  with  nitric  acid  if  necessary,  is  heated,  first  on  a 
water-bath  and  then  over  a  free  flame,  until  coagulation  is  com- 
plete. The  precipitate  is  collected  on  a  small  filter,  and  washed 
with  water,  alcohol,  and  ether.  The  contained  nitrogen  is  now  esti- 
mated according  to  Kjeldahl's  method,  when  the  result  multiplied 
by  6.3  will  indicate  the  corresponding  amount  of  albumin. 

If  it  is  desired  to  estimate  the  amount  of  the  individual  albumins 
separately,  they  are  first  isolated,  as  has  been  described,  and  are  then 
subjected  to  Kjeldahl's  process.  In  this  case,  however,  ammonium 
sulphate  cannot  be  used  for  purposes  of  salting. 

THE  PIGMENTS  OF  THE  URINE. 

Of  the  chemical  nature  of  the  pigments  of  normal  urine  little  is 
known  that  is  definite.  According  to  some  observers,  the  yellow 
color  is  due,  in  part  at  least,  to  tlie  presence  of  so-called  urochrome, 
which  in  turn  is  regarded  as  identical  with  the  normal  urobilin  of 
MacMunn.  Others,  again,  claim  that  there  is  no  reason  to  suppose 
that  a  difference  exists  between  this  normal  urobilin  and  the  urobilin 
of  Jaffe,  which  is  mostly  observed  under  pathological  conditions,  but 
which  may  occur  also  in  health.  Jaffe's  urobilin,  further,  is  held  by 
some  to  be  identical  with  the  hydrobilirubin  which  results  from 
bilirubin  through  the  action  of  sodium  amalgam.  Of  late,  how- 
ever, this  view  has  been  questioned,  especially  as  bilirubin  on  oxida- 
tion furnishes  a  substance,  choletelin,  which  cannot  be  distinguished 
from  hydrobilirubin  on  the  one  hand,  or  urobilin  on  the  other.  A 
similar  pigment,  or  one  which  is  identical  with  urobilin,  has  further 
been  obtained  from  hsematoporphyrin.      That  the  urobilin  which  is 


314  THE   URINE. 

notably  observed  under  pathological  conditions  can  be  formed  within 
the  body  in  the  absence  of  micro-organisms  is  now  a  wcll-cstal)lished 
fact.  AVe  thus  find  that  in  diseases  in  which  the  elimination  of  bile 
through  the  usual  channels  is  prevented,  urobilin  may  occur  in  the 
urine,  nevertheless  ;  and  it  has  further  been  noted  that  both  at  the 
beginning  and  at  the  end  of  jaundice  increased  amounts  are  found. 
Similar  results  have  been  obtained  when  from  any  cause  an  increased 
destruction  of  blood-pigment  occurs.  AVe  may  thus  imagine  that  in 
such  cases  the  urol^ilin  results  from  bilirubin  through  an  extensive 
oxidation  to  choletelin.  This  view  of  the  origin  of  urobilin,  of 
course,  does  not  necessarily  preclude  the  possibility  that  a  certain 
amount  of  the  pigment,  which,  as  I  have  said,  may  normally  also 
occur  in  the  urine,  may  be  derived  from  bilirubin  through  a  process 
of  reduction  in  the  intestinal  tract.  But,  as  is  apparent  from  the 
considerations  just  related,  M-e  are  scarcely  in  a  position  to  speak 
authoritatively  of  the  origin  of  the  normal  urinarv  pigments.  The 
chemical  position  of  the  colorless  mother-sulxstance  of  urobilin,  more- 
over, which  is  spoken  of  as  urobilinogen,  and  which  can  usually  Ije 
demonstrated  whenever  urobilin  also  is  present,  is  thus  far  not  clear. 

According  to  Garrod,  the  urobilin  of  the  urine  is  identical  with 
the  stercobilin  of  the  feces,  both  in  composition  and  properties,  but 
differs  conspicuously  from  hydrobilirubin,  especially  in  the  much 
smaller  percentage  of  nitrogen  which  it  contains,  viz.,  4.11  per  cent., 
as  compared  with  9.22.  The  elementary  composition  of  urobilin  is 
given  by  Garrod  and  Hopkins  as  :  C,  63.58  per  cent. ;  H,  7.84 ; 
X,  4.11,  and  O,  24.47.  Garrod  further  states  that  by  acting  upon 
urochrome  with  acids  he  never  succeeded  in  obtaining  any  prod- 
uct showing  the  uroljilin  band,  or  yielding  the  well-known  fltiores- 
cence  with  zinc  chloride  and  ammonia,  as  Thudichum  claimed. 
But  he  found  that  a  substance  having  both  these  properties  is 
readily  obtained  by  the  action  of  aldehyde  upon  an  alcoholic  solu- 
tion of  urochrome.  In  a  short  time — shorter  still  when  the  liquid 
is  warmed — an  absorption-band  appears  like  that  of  urobilin,  and 
the  tint  of  the  solution  deepens  to  a  rich  orange-yellow.  AVith  zinc 
chloride  and  ammonia  a  brilliant  green  fluorescence  occurs,  and 
the  band  is  shifted  toward  red,  as  that  of  uroljilin  is  under  like 
conditions. 

Isolation  of  Uroclirome. — To  demonstrate  the  presence  of  so-called 
urochrome  in  normal  urine,  the  fluid  is  acidulated  with  1  or  2  c.c. 
of  dilute  sulphuric  acid  pro  liter.  On  filtering,  it  is  saturated  with 
ammonium  sulphate.  The  resulting  precipitate  is  dried  and  ex- 
tracted with  warm  and  slightly  ammoniacal  alcohol.  The  pigment 
passes  into  solution,  and  is  obtained  on  evaporation  of  the  alcohol  as 
an  amorphous  reddish-brown  substance,  which  is  readily  soluble  in 
acidulated  water,  chloroform,  and  common  alcohol,  but  is  practically 
insoluble  in  ether  and  benzol.  According  to  Garrod,  its  solutions 
do  not  give  rise  to  any  bands  of  absorption,  and  do  not  fluoresce 
upon  the  addition  of  ammonia  and  zinc  chloride.     Gautier,  on  the 


THE  PIGMENTS  OF  THE   URINE.  315 

•other  hand,  states  that  its  acidulated  solutions  show  a  band  of 
absorption  about  F,  and  that  the  remainder  of  the  spectrum  from 
about  G  on  to  the  right  is  obscured.  He  adds  that  in  this  respect 
urochrome  and  choletelin  are  alike. 

Garrod  regards  the  action  of  aldehyde  upon  an  alcoholic  solution  of 
nrochrome,  outlined  above,  as  a  very  delicate  test  for  the  pigment. 
The  process  can  bo  stopped  then  h\  simple  dilution  with  water,  as 
aldeliyde  has  no  such  action  upon  aqueous  solutions  of  urochrome. 
If,  however,  the  action  is  allowed  to  continue,  a  further  change 
ensues.  The  liquid  reddens  and  a  second  baud  appears  in  the  violet. 
The  fluorescence  can  still  be  obtained  with  zinc  chloride  and  ammonia, 
and  both  bands  are  shifted  toward  red  and  are  closer  together  than 
before. 

The  term  uroerythrin  has  been  applied  to  the  pigment  which 
imparts  the  salmon-red  color  to  sediments  which  are  composed  of 
urates  or  uric  acid.  Of  its  chemical  nature,  however,  nothing  defi- 
nite is  known,  but  there  is  evidence  to  show  that  it  also  is  a  deriva- 
tive of  the  normal  coloring-matter  of  the  blood.  It  contains 
62.51  per  cent,  of  carbon  and  5.79  per  cent,  of  hydrogen.  Its 
amount  is  noticeably  increased  in  extensive  disease  of  the  liver,  as 
also  in  conditions  associated  with  an  increased  destruction  of  red 
corpuscles. 

Isolation  of  Uroerythrin. — As  has  been  stated,  the  salmon  color  of 
sediments  of  urates  and  uric  acid  is  due  to  this  pigment.  In  their 
absence  the  itrine  is  precipitated  with  neutral  acetate  of  lead  or 
barium  chloride.  If  uroerythrin  is  present  beyond  traces,  it  is  thrown 
down,  and  colors  the  resulting  precipitate  a  more  or  less  intense 
salmon  red.  The  pigment  is  soluble  in  boiling  alcohol,  and  may 
thus  be  extracted.  Its  solutions  are  said  to  give  rise  to  two  bands 
of  absorption  to  the  left  of  F. 

Urobilin. — Urines  which  contain  much  urobilin,  viz.,  the  patho- 
logical urobilin  of  Jaife,  present  a  dark-yellow  color,  which  may  be 
imparted  to  the  foam  on  shaking.  They  are  thus  quite  similar  to 
icteric  urines. 

Tests. — To  identify  the  substance,  the  urine  is  precipitated  with  a 
mixture  of  barium  hydrate  and  barium  chloride.  If  notable  quanti- 
ties of  urobilin  are  present,  the  precipitate  is  thus  colored  a  more  or 
less  intense  brownish-red.  On  boiling  with  acidulated  alcohol  the 
pigment  is  then  extracted,  and  imparts  a  brow^nish  or  pomegranate- 
red  color  to  the  alcoholic  solution  (v.  Jaksch). 

Gerhardt's  test  also  is  very  serviceable.  To  this  end,  10-20  c.c. 
of  urine  are  extracted  with  chloroform  by  shaking.  A  few  drops 
of  a  dilute  solution  of  iodopotassic  iodide  are  added  to  the  extract, 
when,  upon  the  further  addition  of  a  dilute  solution  of  sodium 
hydrate,  the  solution  is  colored  yellow  or  yellowish  brown  and  ex- 
hibits a  beautiful  greenish  fluorescence. 

If  the  substance  cannot  be  demonstrated  with  these  tests,  the 
urine  is  acidulated   with    hvdrochloric    acid   and  allowed  to  stand 


316  THE   URINE. 

exposed  to  the  air,  so  that  any  urobilinogen  that  may  be  present 
is  transformed  into  the  free  pigment.  The  fluid  is  then  examined 
with  the  spectroscope,  when  in  the  presence  of  urobilin  a  distinct 
band  of  absorption  is  obtained  between  b  and  F,  extcndiuG:  beyond 
F  to  the  right.  A  similar  band  is  also  obtained  in  alkaline  solu- 
tion, but  is  not  so  intense  and  does  not  extend  beyond  F. 

Isolation. — To  isolate  the  pigment,  if  present  in  large  amounts, 
the  urine  is  directly  precipitated  with  ammonia  and  chloride  of 
zinc.  The  precipitate  is  thoroughly  washed  with  water,  extracted 
with  alcohol  by  boiling,  dried,  and  then  dissolved  in  ammonia.  The 
resulting  solution  is  precipitated  with  subacetate  of  lead,  the  ])recipi- 
tate  washed  with  water,  and  extracted  with  boiling  alcohol  as  before, 
and  decomposed  with  acid  alcohol.  The  filtered  alcoholic  solution  is 
treated  with  one-half  its  volume  of  chloroform  and  diluted  with 
water ;  the  urobilin  ]->asses  into  the  chloroform  on  moderate  agita- 
tion. The  chloroform  solution  is  then  washed  with  water.  On 
final  distillation  the  pigment  remains  as  an  amorphous  reddish  mate- 
rial, which  can  be  further  purified  by  washing  Avith  ether,  which  takes 
up  contaminating  red  pigments.  The  substance  is  readily  soluble 
in  alcohol,  amyl  alcohol,  and  chloroform,  less  readily  so  in  water 
and  ether. 

Ehrlich's  Reaction. — AVhen  normal  urine  is  treated  with  an 
equal  volume  of  a  saturated  solution  of  sulphanilic  acid  in  5  per 
cent,  hydrochloric  acid  to  which  a  0.5  per  cent,  solution  of  sodium 
nitrite  has  been  added  in  the  proportion  of  1  :  40,  and  the  mixture 
is  then  rendered  strongly  alkaline  with  ammonia,  a  more  or  less  well- 
marked  orange  color  develops.  Under  pathological  conditions,  on 
the  other  hand,  and  notably  in  typhoid  fever,  a  red  color*  results 
which  varies  in  intensity  from  a  light  carmin  to  a  deep  garnet-red. 
This  reaction  is  known  as  Ehrlich's  diazo-reaction,  as  it  is  dependent 
upon  the  presence  of  a  diazo-  compound  of  the  nature  of  diazo-ben- 
zene-sulphonic  acid,  in  the  reagent.  This  results  through  an  inter- 
action between  the  sodium  nitrite  and  the  sulphanilic  acid,  as  repre- 
sented in  the  equations  : 

(1)  NaNO^  +HC1     =HNO.,  -|- NaCl. 

/Nil,  /N=N 

(2)  QH/   I     ■      +  HXO,  =  CeIT,<  I  +  2H,0 

Sulphanilic  Diazo-benzene- 

acid.  sulphonic  acid. 

The  reaction  is  supposedly  referable  to  alloxy-proteinic  acid. 

Under  pathological  conditions  still  other  pigments  may  be  found 
in  the  urine.  These  comprise  hcemoglobin  and  its  derivatives, 
hsematin,  methsemoglobin,  and  hsematoporphyrin  ;  further,  also  uro- 
rubrohtematin  and  urofuscohsematin,  which  are  also  imdoubtedly 
derived  from  haemoglobin,  but  which  have  thus  far  been  found  in 
the  urine  on  only  one  occasion  ;  further,  the  common  pigments  of  the 
bile ;    and,    finally,  substances  which    belong   to  the    class    of  the 


THE  PIGMENTS  OF  THE   URINE.  317 

so-called  melanins.  For  a  consideration  of  the  various  pathological 
conditions  under  which  the  bodies  may  be  met  with,  however,  the 
reader  is  referred  to  special  works  on  diagnosis.  At  this  place  I 
shall  merely  describe  the  more  common  tests  by  which  their 
presence  can  be  demonstrated. 

The  Blood-pigments. — If  the  microscopical  examination  of  the 
urine  shows  the  presence  of  red  blood-corpuscles  in  the  sediment, 
further  chemical  examination  is,  of  course,  unnecessar}'.  Cases  of 
simple  haemoglobinuria,  in  contradistinction  to  hsematuria,  may  occur, 
however,  in  which  dissolution  of  the  haemoglobin  has  taken  place  in 
the  circulation  already,  and  in  which  biood-corpuscles  do  not  appear 
in  the  urine.  In  such  an  event  the  demonstration  of  blood-pig- 
ment can  be  made  only  by  chemical  methods.  Its  presence,  it  is 
true,  is  usually  indicated  by  the  color  of  the  urine,  but  this  may 
be  simulated  by  other  substances  as  well. 

Heller's  Test. — This  is  the  most  convenient  test  for  demon- 
strating the  presence  of  blood-pigment  in  the  urine,  and,  in  the  modi- 
fication here  given,  exceedingly  sensitive.  It  is  based  upon  the 
decomposition  of  the  pigment  in  question  by  means  of  caustic 
alkali  and  the  resulting  formation  of  hsemochromogen.  To  this 
end,  a  small  amount  of  the  urine,  or,  still  better,  of  the  sediment,  is 
rendered  strongly  alkaline  with  caustic  alkali  and  boiled.  On  stand- 
ing, the  precipitated  earthy  phosphates  settle  to  the  bottom,  and  are 
colored  a  more  or  less  intense  carmin  by  the  hsemochromogen,  which 
has  likewise  separated  out.  That  the  pigment  is  in  reality  hsemo- 
chromogen can  be  readily  demonstrated  on  spectroscopic  exami- 
nation (see  page  354),  When  controlled  in  this  manner,  the  test 
is  exceedingly  sensitive,  and  may  still  yield  a  positive  result  even 
when  the  chemical  test  by  itself  does  not  give  a  well-pronounced 
reaction. 

Spectroscopic  Examination. — On  direct  spectroscopic  exam- 
ination the  spectrum  of  methsemoglobin  is  usually  obtained.  The 
urine  should  first  be  acid,  and  if  necessary  a  little  acetic  acid  is 
added.  On  the  addition  of  a  little  ammonia  and  ammonium  sul- 
phide and  subsequent  filtration  the  broad  band  of  haemoglobin  is  then 
obtained.  With  oxyhsemoglobin,  on  the  other  hand,  the  two  bands 
between  D  and  E  are  observed  ;  and  upon  the  subsequent  addition 
of  ammonia  and  ammonium  sulphide  and  filtration  the  spectrum  of 
reduced  haemoglobin  results.  If  this  does  not  appear  distinctly,  the 
solution  is  treated  with  an  excess  of  sodium  hydrate  solution,  and 
will  then  give  the  spectrum  of  hjemochromogen. 

Haematin. — Haematin  is  very  rarely  found  in  the  urine.  Its 
presence  as  such  can  be  determined  only  by  spectroscopic  examina- 
tion. Like  haemoglobin  and  methaemoglobin,  it  gives  Heller's 
reaction. 

Haematoporpliyriii. — According  to  Garrod,  traces  of  haematopor- 
phyrin  may  be  found  in  every  urine.  Larger  quantities  are 
observed  in  a  number  of  diseases,  but  even  in  these  the  amount 


318  THE   URINE. 

is  usually  so  small  that  its  presence  will  scarcely  be  suspected 
from  simple  inspection.  Typical  haematoporphyrinuria,  on  the 
other  hand,  may  be  observed  following  the  prolonged  administra- 
tion of  sulphonal,  trional,  and  tetronal,  or  in  cases  of  poisoning  with 
the  substances  in  question.  The  urine  then  appears  dark  red  in. 
color,  and  on  standing  may  turn  almost  black.  As  Hammarsten 
has  pointed  out,  this  change  in  color  is  only  in  part  due  to  hsemato- 
porphyrin,  and  is  largely  referable  to  other  red  and  reddish-brown 
pigments  of  unknown  character.  Whether  or  not  different  hsemato- 
porphyrins  exist  has  not  been  definitely  determined,  but  is  probable. 
In  freshly  voided  urines  hsematoporphyrin  probably  exists  in  com- 
bination with  some  other,  still  unknown  body,  with  which  it  forms 
a  colorless  chromogen.  From  this  the  free  pigment  then  develops 
on  exposure  to  the  air. 

Like  the  common  blood-pigments  and  hcematin,  hoematoporphyrin 
also  reacts  with  Heller's  test.  To  prove  its  presence,  however,  as 
such,  a  spectroscopic  examination  is  necessary.  To  this  end,  the 
urine  is  ])recipitated  with  barium  hydrate  and  barium  chloride. 
The  precipitate  is  washed  and  allowed  to  stand  in  contact  with 
acidulated  alcohol,  which  extracts  the  pigment.  After  filtering, 
the  solution  is  examined  with  the  spectroscope  ;  if  subsequently 
the  solution  is  rendered  alkaline  with  ammonia,  the  spectrum  of 
hsematoporphyrin  in  alkaline  solution  is  obtained.  To  isolate  the 
substance  as  such,  the  acid  solution  is  mixed  with  a  little  chloro- 
form and  diluted  with  water.  On  gentle  agitation  the  chloroform 
takes  up  the  greater  portion  of  the  hgematoporphyrin,  while  a 
small  fraction  and  other  pigments  remain  in  the  diluted  alcoholic 
solution.  On  evaporating  the  chloroform  extract  the  substance  is 
obtained  in  comparatively  pure  form. 

Neumeister  states  that  besides  ha?matoporphyrin  another  deriva- 
tive of  the  blood-pigment  may  be  observed  in  cases  of  poisoning 
with  sulphonal,  which,  in  contradistinction  to  the  first,  contains  iron. 
This  does  not  react  with  Heller's  test,  however,  while  the  color 
of  the  urine  is  the  same  as  in  typical  haematoporphyrinuria.  The 
pigment  is  precipitated  by  an  alkaline  barium  chloride  solution, 
and  can  be  subsequently  dissolved  in  acid  alcohol.  This  solution 
presents  a  reddish-violet  color,  and  shows  one  broad  band  of  absorp- 
tion in  the  blue  portion  of  the  spectrum  immediately  bordering  on 
the  green. 

On  rendering  the  solution  alkaline  with  ammonia  the  pigment  is 
thrown  down.  On  adding  an  excess  of  sodium  hydrate  solution,  on 
the  other  hand,  it  is  dissolved,  while  the  liquid  assumes  a  yellow 
color.  This  solution  then  shows  a  sharp,  narrow  line  in  the  green, 
near  the  blue  portion  of  the  spectrum,  but  disappears  after  the 
solution  has  stood  for  some  time. 

Urorubrohsematin  and  Urofuscolisematin. — These  pigments  were 
isolated  by  Baumstark  from  the  urine  of  a  leprosy  patient,  but  have 


THE  PIGMENTS  OF  THE   URINE.  319 

not  been  encountered  since.     Their  relation  to  hrematin  is  apparent 
from  the  formula; : 

C32H32N^04Fe,  hfematin  (Nencki  and  Sieber). 

Cs^HgjN^OjFe,  ha?matin  (Hoppe-Seyler). 

CggHg^NgOsoFe,  urorubrohsematin. 

C68Hi06^8O26)  urofusoolisematin. 

The  pigments  were  isolated  as  follows  :  the  urine,  which  presented 
a  color  varying  from  a  dark  red  to  a  brownish  red,  was  dialyzed  and 
the  final  contents  of  the  dialyzer  dissolved  in  sodium  hydrate  solu- 
tion ;  upon  the  addition  of  hydrochloric  acid  to  this  solution 
urofuscohsematin  separated  out  in  brown  flakes,  while  the  second 
pigment  remained  in  solution,  coloring  this  a  beautiful  red.  After 
filtering  off  the  first,  the  solution  was  again  dialyzed,  when  the 
second  pigment  separated  out. 

Whether  or  not  any  relation  exists  between  these  two  bodies  and 
hsematoporphyrin  in  impure  form,  as  Hammarsten  suggests,  must 
remain  an  open  question. 

Melanins. — Notably  in  association  with  the  existence  of  melanotic 
tumors,  but  at  times  also  in  other  diseases  which  are  associated  with 
an  increased  destruction  of  red  blood-corpuscles,  urines  are  met  with 
which  gradually  turn  a  dark  brown  or  black  on  standing.  When 
freshly  voided,  however,  they  commonly  present  a  normal  color. 
The  pigment  or  pigments  which  are  thus  formed  belong  to  the  class 
of  melanins,  and  are  identical  with  those  which  can  be  obtained 
from  the  pigmented  growths.  They  are  probably  eliminated  in 
combination  with  some  other  substance  which  is  as  yet  unknown,  as 
colorless  meJanogens  and  from  which  the  free  pigments  are  obtained 
on  oxidation.  They  are  unquestionably  derived  from  the  common 
pigments  of  the  blood,  but  are  individually  little  knoAvn. 

To  prove  that  the  change  in  the  color  of  the  urine  is  referable  to 
melanins,  a  fresh  specimen  should  be  procured,  and  treated  with 
bromine-water.  If  the  chromogens  in  question  are  present,  the 
resulting  precipitate,  which  is  yellow  at  first,  turns  black  on  standing. 
On  the  addition  of  a  few  drops  of  a  strong  ,  solution  of  ferric 
chloride  a  similar  reaction  is  obtained. 

To  isolate  the  pigments  from  the  urine,  the  fluid  is  first  precipi- 
tated with  an  alkaline  solution  of  barium  chloride.  From  the 
resulting  precipitate  the  pigments  are  extracted  with  a  concentrated 
solution  of  sodium  carbonate,  and  are  then  precipitated  by  adding 
an  excess  of  sulphuric  acid.  By  redissolution  in  a  dilute  solution 
of  sodium  hydrate  and  reprecipitation  with  acetic  acid  they  can  be 
obtained  in  comparatively  pure  form.  But  it  will  be  noted  that  a 
certain  fraction  remains  in  the  acetic  acid  solution,  which  indicates 
the  existence  of  at  least  two  different  pigments.  The  soluble 
form  has  been  termed  phymatorhusin,  and,  according  to  Nencki 
and   Sieber,   contains   no  iron,  while   Moruer   claims    that    this    is 


From  urine. 

55.76  per 
5.95  '• 

cent. 

12.27   " 

a 

9.01  " 

u 

0.20  " 

" 

320  THE   URISE. 

present.     Elementary  analysis  of  tiiis  pigment  has  given  the  fol- 
lowing results  (Mdrner) : 

From  growth. 

Carbon       55.32  to  56.13  per  cent. 

Hydrogen 5.65  to    6. 33   "      '" 

Nitrogen 12.30  " 

Sulphur 7.97  "      " 

Iron 0.063  to  0.081   "      " 

From  melanotic  growths  in  horses  a  hippomelanin  has  been 
obtained,  which,  in  contradistinction  to  the  first,  is  soluble  in  solu- 
tions of  the  alkalies  with  difficulty. 

The  Bile -pigments. — Bile-pigments  are  never  found  in  the 
urine  under  normal  conditions.  As  a  rule,  freshly  voided  urine 
contains  only  bilirubin.  If  a  complicating  cystitis,  however,  exists, 
the  common  derivatives  of  bilirubin,  viz.,  biliverdin,  bilifuscin, 
biliprasin,  and  bilihumin,  may  also  be  encountered. 

Bile-containing  urines  present  a  very  characteristic  color,  which 
may  vary  from  a  bright  golden-yellow  to  a  greenish  brown,  and  on 
microscopical  examination  it  is  common  to  find  the  morphological 
elements  stained  an  intense  yellow.  This  color  is  further  imparted 
to  the  foam  on  shaking.  But  as  urobilin  when  present  in  large 
amounts  may  impart  a  similar  color  to  the  urine,  it  is  always 
better  to  resort  to  chemical  tests.  These  have  been  descril)ed  in 
detail  in  the  section  on  the  Bile,  and  are  directly  applicable  also  to 
the  urine  (.see  page  175). 

Other  pigments  also  may  occur  in  the  urine  after  the  ingestion  of 
various  drugs,  but  as  the  products  thus  formed  are  of  no  special 
interest  from  the  standpoint  of  animal  chemi.stry,  they  are  not  con- 
sidered at  this  place. 

The  Bile -acids. — The  occurrence  of  bile-acids  in  the  urine  is 
solely  a  pathological  phenomenon.  In  normal  urines  they  are  never 
found,  and  even  in  complete  obstruction  of  the  common  duct  their 
amount  is  quite  small.  To  demonstrate  their  presence,  they  must 
first  be  i.solated  as  Platner's  bile,  and  can  then  be  identified  by 
polarimetric  examination,  their  action  upon  the  frog's  heart,  etc. 
(see  page  163). 

Fats,  Cholesterin,  and  Lecithins. 

Fats. — Traces  of  fat  may  be  observed  in  the  urine  under  normal 
conditions  when  excessive  amounts  have  been  ingested.  Diiring 
pregnancy  also  a  lipuria  has  been  noted,  and  is  probably  associated 
with  the'  development  of  the  function  of  lactation.  Otherwise  the 
condition  is  essentially  a  pathological  phenomenon,  and  is  notably 
observed  in  acute  yellow  atrophy,  in  phosphorus  poisoning,  follow- 


THE  PIGMENTS  OF  THE    URINE.  321 

ing  fracture  of  the  long  bones,  etc.  The  largest  amounts,  however, 
are  found  in  cases  of  so-called  chyluria.  Owing  to  the  existence  of 
the  fats  in  fine  emulsions,  such  urines  may  resemble  milk  in  their 
general  appearance,  and  on  standing  a  layer  of  ''  cream "  forms  at 
the  top. 

To  establish  the  presence  of  fats,  the  surface  layer  of  the  urine  is 
extracted  with  ether,  the  ether  is  evaporated,  and  the  residue  brought 
in  contact  with  a  piece  of  paper,  when  characteristic  stains  result. 

Cholesterin. — Cholesterin  is  very  rarely  found  in  the  urine,  and 
has  thus  far  been  encountered  only  under  pathological  conditions. 
It  probably  always  occurs  in  crystalline  form,  and  is  thus  readily 
recognized.  If  any  doubt  exists,  the  substance  in  question  is 
examined  as  has  been  described  (page  180). 

Lecithins. — Lecithins  as  such  have  been  observed  in  the  urine 
only  in  chyluria,  where  they  are  commonly  present  in  association 
with  cholesterin  and  fat.  One  of  the  derivatives  of  lecithin,  how- 
ever, is  found  also  in  the  urine  under  normal  conditions,  though 
in  very  small  amounts.  This  is  glycerin-phosphoric  acid.  It  is  no 
doubt  referable  to  decomposition  of  the  lecithins  of  the  food  in  the 
intestinal  canal,  but  may  at  times  also  be  derived  from  the  lecithins 
of  the  tissues.  To  demonstrate  its  presence,  several  liters  of  urine 
are  freed  from  the  common  phosphates  by  rendering  the  urine 
alkaline  with  barium  hydrate  and  precipitating  the  heated  mixture 
with  barium  chloride.  The  excess  of  barium  is  removed  with 
carbonic  acid  and  the  filtrate  evaporated  to  a  syrup.  This  is  ex- 
tracted with  absolute  alcohol,  when  the  remaining  material  is  dis- 
solved in  a  little  water  and  boiled  with  hydrochloric  acid.  The 
glycerin-phosphoric  acid  is  thus  decomposed,  with  the  liberation  of 
glycerin  and  phosphoric  acid.  On  evaporating  to  dryness,  the 
residue  is  extracted  with  water,  and  the  presence  of  phosphoric 
acid  demonstrated  in  the  aqueous  solution  by  the  usual  tests. 

Ferments. 

Every  urine  contains  ferments  which  are  thought  to  be  identical 
with  the  pepsin,  ptyalin,  and  chymosin  of  the  digestive  fluids.  It 
can  be  shown,  as  a  matter  of  fact,  that  substances  are  present 
which  are  capable  of  digesting  fibrin  in  acid  solution,  of  inverting 
starch  to  maltose,  and  of  coagulating  milk.  There  is  no  proof, 
however,  that  these  bodies  are  derived  from  the  digestive  glands, 
as  Neumeister  and  others  claim. 

Gases. 

In  normal  urine  a  certain  amount  of  oxygen,  nitrogen,  and  nota- 
bly of  carbon  dioxide  is  found  in  solution,  and  can  be  withdrawn 
by  the  air-pump.  Under  pathological  conditions,  further,  a  variable 
amount  of  hydrogen  sulphide  may  be  encountered.  Tliis  notably 
occurs  in   cases  of  cystitis,  in  which   the  decomposition  of  albumin 

21 


322  THE  URINE. 

ami  sulphur  bodies  may  already  take  place  within  the  bladder,  owing 
to  the  activity  of  various  micro-organisms.  But  in  a  few  isolated 
cases  the  gas  was  apparently  derived  from  the  intestinal  tract,  and 
absorbed  either  directly  from  the  rectum  or  indirectly  from  the 
blood. 

All  urines  when  exposed  to  the  air  sooner  or  later  contain  hydro- 
gen sulphide  in  the  free  state,  which  is  referable,  as  stated  above, 
to  the  action  of  certain  micro-organisms.  Especially  large  amounts 
are  observed  when  cystin-containing  urines  are  thus  allowed  to 
undergo  decomposition.  To  test  for  hydrogen  sulphide,  a  strip  of 
filter-paper  is  moistened  with  a  few  drops  of  a  solution  of  sodium 
hydrate  and  one  of  lead  acetate,  and  is  then  clamped  in  the 
neck  of  the  bottle  containing  the  urine.  If  the  gas  in  question  is 
present,  the  paper  is  colored  a  grayish  brown  or  black,  owing  to 
the  formation  of  lead  sulphide.  When  present  in  large  amounts 
it  is  detected  also  by  its  odor. 

Ptomains. 

So  far  as  known,  ptomains  are  not  found  in  the  urine  under 
normal  conditions.  In  disease,  however,  various  basic  substances 
have  been  encountered  which  supposedly  belong  to  this  class.  But 
with  the  exception  of  cadaverin  and  putrescin,  which,  as  has  been 
stated,  may  occur  in  association  with  cystinuria,  these  bodies  have 
been  isolated  in  amounts  scarcely  sufficient  to  establish  their 
chemical  nature.  This  holds  good  more  especially  of  the  bodies 
which  Griffith  claims  to  have  isolated  from  the  urine  of  patients 
suffi'ring  from  scarlatina,  measles,  mumps,  carcinoma,  etc. 

As  regards  the  origin  of  putrescin  and  cadaverin  in  cystinuria,  the 
opinion  prevails  that  they  are  due  to  a  specific  form  of  intestinal 
putrefaction.  This  is,  however,  not  necessarily  the  case,  and  in  my 
opinion  the  diaminuria,  like  the  cystinuria,  is  the  expression  of  a 
distinct  metabolic  disturbance.  I  have  pointed  out  that  both  diamins 
can  be  derived  from  arginin  and  lysin  in  the  laboratory,  and  there 
is  every  reason  to  suppose  that  the  same  transformation  can  also 
occur  in  the  living  organism.  That  arginin  at  least  actually  occurs 
in  the  tissues  of  the  body  has  been  demonstrated  by  Gulewitch,  who 
found  the  substance  in  the  spleen. 

The  quantity  of  the  diamins  which  may  be  eliminated  in  the 
urine  in  cases  of  cystinuria  is  quite  variable.  On  some  days  traces 
only  or  none  at  all  is  found,  while  at  other  times  very  considerable 
amounts  may  be  obtained.  In  one  of  my  cases  I  was  able  to  isolate 
1.6  grammes  of  the  benzoylated  cadaverin  from  the  collected  urine 
of  twenty-four  hours. 

To  demonstrate  the  presence  of  diamins,  the  method  of  Bau- 
raann  and  v.  Udranszky  is  most  conveniently  employed.  To  this 
end,  the  collected  urine  of  twenty-four  hours  or  mure  is  benzoylated 
by  shaking  with  benzt)yl  chloride  in  the  presence  of  sodium  hydrate. 


THE  PTOMAINES  OF  THE    URINE.  323 

As  a  general  rule,  25  c.c.  of  the  chloride  and  200  c.c.  of  a  10  per 
cent,  solution  of  sodium  hydrate  are  used  for  1500  c.c.  of  the  urine. 
The  resulting  precipitate,  which  contains  the  earthy  phosphates,  the 
benzoylated  carbohydrates  which  are  normally  present  in  every 
urine,  and  the  greater  portion  of  the  benzoylated  diamins,  is  then 
filtered  oif,  extracted  with  boiling  alcohol,  filtered,  and  the  alcoholic 
extract  concentrated  on  a  water-bath.  This  solution  is  then  poured 
into  thirty  times  its  volume  of  water.  On  standing,  the  benzoy- 
lated diamins  separate  out  in  crystalline  form,  and  are  then  freed 
from  adhering  carbohydrates  by  repeated  solution  in  alcohol  and 
precipitation  in  water.  The  process  is  continued  until  the  desired 
degree  of  purity  is  obtained.  The  resulting  crystals  are  finally 
filtered  off,  dried  over  sulphuric  acid,  and  identified  by  their 
melting-point  and  the  contained  amount  of  nitrogen.  If  both 
diamins  are  present,  the  crystals  lose  their  water  of  crystalliza- 
tion at  120°  C,  and  melt  at  140°  C.  To  separate  them  from 
each  other,  they  are  dissolved  in  a  little  warm  alcohol,  and  are 
treated  with  twenty  times  as  much  ether.  Benzoyl  jjutrescin  is 
thus  thrown  down,  while  the  cadaverin  compound  remains  in  solu- 
tion. The  crystals  of  the  former  melt  at  175°-176°  C,  while  the 
melting-point  of  the  latter  lies  between  129°  and  130°  C. 

A  small  portion  of  the  diamins  remains  in  the  first  filtrate.  To 
isolate  these,  the  liquid  is  acidified  with  sulphuric  acid  and  ex- 
tracted with  ether.  The  ethereal  extract  is  evaporated,  and  the 
final  solution,  before  congealing,  placed  in  as  nuicli  of  a  12  per 
cent,  solution  of  sodium  hydrate  as  is  required  for  its  neutralization. 
From  three  to  four  times  as  much  of  the  alkali  solution  is  then 
added.  On  standing  in  the  cold,  sodium  benzoyl-cystin  separates 
out,  together  with  the  benzoylated  diamins.  The  crystals  are  fil- 
tered otf  and  placed  in  cold  water.  This  dissolves  the  cystin  com- 
pound, while  the  diamins  remain  undissolved.  They  are  soluble  in 
warm  alcohol,  and  can  then  be  separated  from  each  other,  as  has 
been  described. 


CHAPTER    XIII. 

THE  ANIMAL  CELL. 

The  cell  constitutes  the  morphological  unit  of  all  animal  and 
vegetable  life,  and  as  such  is  capable  of  manifesting  those  peculiar 
activities  which  we  regard  as  characteristic  of  living  matter.  In 
its  simplest  form  it  represents  a  tiny  bit  of  a  more  or  less  granular, 
gelatinous  substance — the  so-called  protoplasm — in  the  interior  of 
which  a  somewhat  more  solid-looking  body  can  be  made  out,  which 
is  termed  the  nucleus.  Such  simple  cells  exist  in  nature,  either  as 
such  or  as  conglomerations  of  many  cells  which  represent  the  higher 
forms  of  animal  and  vegetable  life.  All  living  matter,  however, 
whether  simple  or  complex,  has  for  its  origin  the  single  cell.  But 
while  in  the  lowest  forms  of  life  the  single  cell  is  capable  of  per- 
forming all  those  functions  which  are  characteristic  of  living  matter 
by  itself,  we  find,  as  we  ascend  in  the  scale  of  animal  and  vegetable 
life,  that  certain  groups  of  cells  are  here  set  aside  for  the  purpose 
of  executing  separate  functions.  Such  groups  of  cells  we  speak  of 
as  tissues,  and  we  accordingly  find  in  the  highly  organized  mammal 
a  differentiation  of  the  entire  body  into  tissues,  which  according 
to  their  functions  may  be  grouped  as  tissues  of  locomotion,  of  re- 
production, of  digestion,  of  excretion,  etc.  With  such  a  differentia- 
tion of  cells  into  tissues,  however,  the  original  aspect  of  the  cell  is 
more  or  less  changed.  The  highly  differentiated  voluntary  muscle- 
cell  would  thus  at  first  sight  scarcely  be  recognized  as  being  in  any 
way  related  to  the  oval  cell  from  which  it  is  primarily  derived. 
On  careful  examination,  however,  we  find  that,  no  matter  how 
unlike  its  ancestral  cell  such  a  specialized  cell  may  appear,  the  dif- 
ference is  only  apparent,  for  all  cells  of  the  body  consist  at  one 
time  of  their  existence  at  least  of  protoplasm  and  nucleus.  The 
striated  portion  of  the  muscle-cell  is  thus  nothing  more  than  the 
protoplasm  of  the  original  cell,  differentiated  and  modified  in  accord- 
ance with  the  function  which  the  cell  is  to  perform.  In  some  cells, 
however,  such  as  those  of  the  adipose  tissue,  the  original  differentia- 
tion into  protoplasm  and  nucleus  is  apparently  lost,  and  on  ordinary 
microscopical  examination  it  appears  that  such  cells  are  nothing  but 
large  globules  of  fat.  But  with  special  methods  of  staining  we  can 
demonstrate  even  here  that  there  are  a  nucleus  and  protoplasm.  The 
only  cells,  in  fict,  in  which  a  nucleus  cannot  always  be  demonstrated 
are  the  red  corpuscles  of  the  circulating  blood  of  man  and  the 
anthropoid  apes.     We  find,   however,   that  even  in  adult  man  all 

324 


THE  ANIMAL   CELL.  325 

red  corpuscles  at  one  period  of  their  existence,  viz.,  in  their  juve- 
nile form,  are  nucleated,  and  that  under  certain  conditions,  as  after 
copious  hemorrhages,  such  nucleated  corpuscles  may  occur  in  the 
circulating  blood  in  large  numbers.  In  the  bone-marrow,  M'here 
they  are  apparently  formed,  they  are  always  present. 

As  all  manifestations  of  life  are  intimately  associated  with 
chemical  changes  which  bring  about  a  transformation  of  potential 
into  kinetic  energy,  such  changes  must  of  necessity  occur  in  every 
living  cell.  These  changes,  moreover,  must  vary  with  the  func- 
tion which  the  cell  is  to  perform,  and  will  hence  diifer,  to  a 
certain  extent  at  least,  with  the  different  tissues  of  the  complex 
organism.  In  the  monocellular  organisms,  where  all  the  various 
functions  are  performed  by  the  single  cell,  all  these  varying  changes 
must  hence  be  represented.  But  it  is  to  be  inferred  that  in  accord- 
ance with  the  greater  simplicity  in  structure  the  chemical  changes 
also  must  be  of  a  simpler  character.  We  should  hence  expect  that  a 
study  of  the  chemical  processes  which  take  place  in  such  low  forms 
of  life  would  furnish  us  with  a  better  insight  into  the  metabolism 
of  the  complex  organism  than  could  be  attained  from  an  investiga- 
tion of  the  higher  forms.  Unfortunately,  however,  this  is  almost 
an  impossibility  with  the  usual  chemical  and  physical  methods,  for 
in  attempting  such  a  study  we  are  met  with  a  most  serious  obstacle, 
viz.,  our  inability  to  maintain  the  life  of  the  individual  cell  during 
such  an  investigation.  We  would  consequently  have  no  proof  that 
those  products  which  we  could  isolate  from  the  dead  cells  were 
present  as  such  in  the  living  organism.  The  technical  difficulties, 
moreover,  which  stand  in  the  way  of  such  a  study  are  almost  insur- 
mountable. With  microchemical  methods,  it  is  true,  something 
more  definite  might  be  accomplished,  and  although  this  branch  of 
investigation  is  still  in  its  infancy,  it  has  already  furnished  us  with 
a  certain  amount  of  valuable  information.  The  great  advances 
which  have  thus  been  made  in  our  knowledge  of  the  structure 
of  cells  have  largely  been  accomplished  in  this  manner.  Upon 
the  chemical  processes  themselves,  however,  which  take  place 
in  the  cell,  not  much  light  has  as  yet  been  thrown  in  this 
manner.  We  are  consequently  dependent  for  our  knowledge  of 
the  metabolic  processes  which  take  place  in  the  living  body  upon 
a  study  of  the  individual  tissues  as  such,  and  the  changes  which 
result  in  certain  substances  when  introduced  from  without.  An 
analysis  of  these  tells  us  in  what  form  the  various  food-stuffs  are 
represented  in  the  individual  cells.  By  then  studying  the  vari- 
ous decomposition-pn)ducts  which  can  be  isolated  from  the  tissues, 
we  can  in  a  measure  form  an  idea  of  the  manner  in  which  these 
products  were  produced  and  of  the  form  in  which  they  were 
represented  in  the  original  and  more  complex  molecule.  With 
some  tissues,  however,  this  is  more  difficult  than  with  others.  The 
most  satisfactory  results,  on  the  whole,  regarding  the  chemical 
structure  of  the  individual  cell  have  thus  far  been  obtained  from 


326  THE  ANIMAL   CELL. 

an  investigation  of  those  tissues  which  are  especially  rich  in  cells, 
and  in  which  tlie  cells  can  be  more  or  less  completely  separated  from 
the  underlying  matrix  and  from  other  components  which  may  be 
present  at  the  same  time.  This  is  especially  true  of  the  leucocytes 
of  the  blood.  As  these  bodies,  moreover,  are  but  little  differentiated, 
they  may  well  serve  as  types-  of  primitive  cells.  They  are  all 
nucleated,  and  contain  a  varying  amount  of  protoplasm,  which  in 
some  is  capable  of  progressive  movement.  A  limiting  membrane, 
as  in  most  animal  cells,  does  not  exist.  But  it  is  generally  supposed 
that  a  meshwork  of  fine  fibrils  pervades  the  protoplasm,  and  that  in 
the  meshes  a  more  liquid  portion  is  contained.  This  is  termed  the 
hyaloplasm,  in  contradistinction  to  the  more  solid  spongioplasm.  In 
some  forms  the  protoplasm  is  apparently  perfectly  homogeneous,  while 
in  others  it  is  studded  with  numerous  granules  of  variable  size,  which 
execute  more  or  less  active  oscillatory  movements,  which  are  spoken 
of  as  the  molecular  movements  of  Brown. 

The  reaction  of  the  protoplasm  is  alkaline,  while  the  nucleus 
apparently  contains  no  free  alkali.  This  may  be  shown  by  staining 
dried  blood  films  with  a  solution  of  acid  erythrosin  in  chloroform, 
when  it  will  be  seen  that  the  body  of  the  cell  is  colored  a  bright  red, 
while  the  nucleus  is  not  stained.  The  most  intense  reaction  is 
obtained  with  the  protoplasm  of  the  so-called  lymphocytes. 

The  granules  which  are  found  in  certain  forms  of  leucocytes  are 
apparently  of  an  albuminous  nature.  According  to  their  affinity  for 
acid,  basic,  or  neutral  dyes,  they  are  termed  oxyphilic,  basophilic, 
and  neutrophilic,  respectively.  Fatty,  mineral,  or  pigment  granules, 
which  may  be  found  in  other  animal  cells,  are  usually  not  seen  in 
leucocytes.  In  the  eosinophilic  leucocytes,  however,  the  presence  of 
iron  can  readily  be  demonstrated  by  microchemical  methods.  In 
another  form  it  seems  to  be  present  in  all  varieties  of  cells,  and  is 
especially  abundant  in  the  nuclei. 

In  the  mineral  ash  we  further  find  potassium,  sodium,  calcium, 
magnesium,  phosphorus,  and  chlorine,  and  it  is  to  be  noted  that,  in 
contradistinction  to  the  animal  fluids,  the  cell  contains  a  relatively 
larger  amount  of  potassium  and  phosphorus,  while  sodium  and 
chlorine  are  more  abundant  in  the  fluids.  That  the  jihosphates  are 
of  prime  importance  in  the  life  of  the  cell  is  now  definitely 
established,  and  Loew  showed  that  in  the  spirogyra,  for  example, 
growth  and  cellular  division  are  greatly  interfered  with  by  their 
absence.  The  importance  of  the  phosphates  is  without  doubt  con- 
nected with  the  presence  of  the  nucleins  in  the  nuclei — /.  e.,  of 
albuminous  substances  which,  as  we  have  seen,  contain  a  relatively 
large  amount  of  phosphorus  in  organic  combination. 

The  protoplasm  of  the  cell  is  very  rich  in  water,  and,  in  addition 
to  small  amounts  of  mineral  salts,  consists  essentially  of  albumins. 
Some  of  these  are  albumins  proper,  but  the  greater  portion  by 
far  is  represented  by  substances  which  belong  to  the  proteid 
group.     It  appears,  moreover,  that  the  traces  of  serum-albumin  and 


THE  ANIMAL   CELL.  327 

globulin  which  are  j)resent  do  not  represent  integral  constitnents  of 
the  living  proto])lasni,  but  are  merely  to  be  regarded  as  food-stuffs, 
or  possibly  even  as  decomposition-products  of  the  protoplasmic 
molecule. 

Of  other  constituents  of  the  protoplasm,  lecithin  is  the  most 
constant.  In  addition  we  find  certain  protagons,  glycogen,  choles- 
terins,  and  in  dead  cells  also  paralactic  acid,  to  which  the  acid 
reaction  of  dead  protoplasm  is  due. 

The  total  amount  of  solids,  including  the  mineral  salts,  which  are 
thus  found  in  protoplasm,  is  always  small,  and  probably  never 
exceeds  15-20  per  cent.,  while  the  remaining  weight  is  referable  to 
water.  In  some  instances  indeed  almost  the  entire  cell  is  taken  up 
by  the  nucleus,  and  in  the  lymphocytes,  for  example,  only  1.76 
per  cent,  of  the  total  79.21  per  cent,  of  albumins,  as  calculated  for 
the  dry  material,  is  present  in  the  protoplasm. 

The  nucleus  may  be  regarded  as  the  essential  living  part  of  all 
animal  and  vegetable  cells,  and  from  it,  no  doubt,  the  various  func- 
tions of  the  cell  as  a  whole  are  directed.  It  is  intimately  connected 
with  the  process  of  reproduction,  and  during  this  process  it  under- 
goes a  series  of  most  remarkable  changes,  w'hich  are  collectively 
termed  the  karyokinesis  or  karyomitosis  of  the  nucleus.  Micro- 
scopically the  quiescent  nucleus  represents  a  round  or  oval  little 
body,  which  usually  occupies  an  excentric  position  within  the  cell. 
It  is  surrounded  by  a  nuclear  membrane,  and  contains  a  meshwork 
of  extremely  fine  fibrils,  and  one  or  more  nucleoli.  Both  fibrils  and 
nucleoli  possess  a  marked  affinity  for  anilin  dyes,  while  the  nuclear 
membrane  and  the  more  liquid  hyaloplasm  within  the  nuclear 
meshes  are  scarcely  stained  at  all.  We  therefore  recognize  in  the 
nucleus  the  existence  of  chromatic  and  achromatic  substances,  which 
are  usually  spoken  of  as  the  nuclear  chromatins  and  achromatins. 
During  the  process  of  division  a  peculiar  spindle-like  body  is  also 
observed  in  the  nucleus,  which,  like  the  nuclear  hyaloplasm,  is 
achromatic. 

In  contradistinction  to  the  cellular  protoplasm,  the  nucleus  con- 
tains a  much  larger  quantity  of  solids,  but  here  as  there  the  albu- 
mins stand  in  the  foreground.  Whether  or  not  native  albumins 
also  occur  in  the  nucleus  is  not  definitely  known,  but  it  is 
generally  assumed  that  this  is  not  the  case.  The  proteids,  on  the 
other  hand,  are  abundant  and  largely  represented  by  nucleopro- 
teids.  They  are  thought  to  constitute  the  greater  portion  of  the  chro- 
matic constituents  of  the  nucleus,  and  among  them  the  so-called  jj/as^m 
apparently  occupies  a  prominent  position.  This  substance,  while 
not  definitely  known,  is  usually  classed  as  a  nucleoproteid,  but  dif- 
fers from  the  more  commom  forms  in  being  soluble  with  greater 
difficulty.  Especially  abundant  also  is  a  nucleoproteid,  which  Kos- 
sel  and  Lilienfeld  first  obtained  from  the  thymus  gland  of  the  calf. 
This  they  termed  nucleohiston,  from  the  fact  that  on  treatment  with 
hydrochloric  acid  it  is  supposedly    decomposed   into  a   nuclein — 


328  THE  ANIMAL   CELL. 

leuconuclein,  and  histon.  Bang,  however,  denies  that  this  nucleo- 
histon  is  a  true  nucleohiston.  He  regards  it  as  an  albuminate,  and 
states  that  he  could  not  demonstrate  a  nucleinate  of  histon  in  the 
leucocytes. 

Whether  lecithins,  protagon,  and  glycogen,  which  are  constantly 
found  in  the  cellular  protoplasm,  likewise  occur  in  the  nucleus,  is 
not  known. 

Of  mineral  constituents,  iron  is  constantly  present,  and  appar- 
ently occurs  in  combination  with  the  nucleins  in  organic  form. 

Justus  has  further  shown  that  every  nucleus  normally  contains 
iodine. 


CHAPTER    XIV. 

THE  BLOOD. 

General  Considerations. — The  blood  of  man  and  of  almost  all 
vertebrate  animals  is  a  slightly  viscid,  somewhat  opaque-looking 
fluid,  which,  according  to  its  origin  from  an  artery  or  a  vein,  pre- 
sents a  color  that  varies  from  a  bright  scarlet  to  a  dark  bluish-red. 
On  microscopical  examination  it  is  seen  to  contain  a  large  number 
of  cellular  elements,  which  are  partly  colored  and  partly  colorless. 
The  former,  which  greatly  predominate,  are  the  red  corpuscles,  or 
erythrocytes  of  the  blood.  In  man  they  are  homogeneous,  normally 
non-nucleated,  circular,  biconcave  disks,  measuring  on  an  average 
7.5  fx  in  diameter.  When  viewed  through  the  microscope  they  are 
of  a  faint  greenish-yellow  color,  while  en  masse  they  present  the 
ordinary  color  of  the  blood.  The  colorless  bodies,  or  leucocytes,  on 
the  other  hand,  are  all  nucleated  and  in  part  capable  of  executing 
amoeboid  movements.  Some  of  them  are  of  about  the  same  size 
as  the  red  corpuscles,  while  others  are  larger.  The  nucleus  may 
be  single  or  multiple,  and  it  will  be  noted  that  in  the  mononuclear 
forms  the  surrounding  protoplasm  is  more  or  less  homogeneous, 
while  in  the  polynuclear  varieties  it  is  distinctly  granular.  The 
total  number  of  the  leucocytes  per  cubic  millimeter  varies  under 
normal  conditions  between  3000  and  10,000,  and  is  thus  much 
smaller  than  the  number  of  the  red  corpuscles,  w^hich  is  generally 
placed  at  between  4,000,000  and  5,000,000  in  the  same  volume  of 
blood. 

In  addition  to  the  red  corpuscles  and  the  leucocytes,  we  further 
find  a  large  number  of  minute  colorless  disks,  measuring  less  than 
one-half  the  diameter  of  a  red  corpuscle,  and  usually  occurring  in 
bunches  of  from  six  to  a  dozen  or  more.  They  are  termed  the 
plaques  or  blood-plates  of  Bizzozero.  On  an  average,  about  635,000 
are  found  in  the  cbmm.  Other  morphological  elements  are  not 
found  in  the  blood  under  normal  conditions,  while  in  disease  nucleated 
red  corpuscles,  both  of  the  adult  and  the  embryonic  type,  as  well  as 
other  forms  of  leucocytes,  may  be  encountered. 

When  blood  is  drawn  from  the  living  body  and  is  allowed  to 
stand,  it  wall  be  noted  that  after  a  variable  length  of  time  the  entire 
mass  is  transformed  into  a  semisolid,  jelly-like  material,  which  is 
termed  the  placenta  sanguinis  or  blood-clot.  On  microscopical  ex- 
amination this  will  be  seen  to  consist  of  a  dense  network  of  fibres, 
in  the  meshes  of  which  the  corpuscles  of  the  blood  are  found.  If 
the  clot  is  now  carefully  separated  from  the  walls  of  the  vessel,  it 

329 


330  THE  BLOOD. 

undergoes  slirinkage,  and  presses  out  from  its  meshes  a  clear,  straw- 
colored  fluid,  which  is  termed  the  blood-serum.  Tiiis  gradually 
increases  in  amount,  while  the  size  of  the  clot  diminish(!S,  and  may 
then  be  utilized  for  the  purpose  of  chemical  examination.  The 
fibrous  material  which  is  formed  during  the  process  of  clotting  is 
termed  fibrin.  Its  formation  is  intimately  associated  with  the  death 
of  the  organism,  either  local  or  general,  and  is  dependent  in  the  first 
instance  upon  the  presence  of  a  specific  ferment — the  so-called  fibrin 
ferment,  or  thrombin  of  Alexander  Schmidt.  In  the  circulating 
blood  fibrin  is  not  found,  but  we  here  meet  with  its  mother-substance, 
fibrinogen,  which  is  not  present  in  the  blood-serum.  The  blood- 
plasma,  viz.,  the  fluid,  non-cellular  portion  of  the  circulating  blood, 
thus  differs  from  the  blood-serum  in  containing  fibrinogenic  material, 
but  not  the  fibrin  ferment,  which  is  found  in  the  serum.  The  fer- 
ment itself  results  from  decomposition  of  the  cellular  elements  of 
the  blood,  notably  of  the  blood-plates.  This  may  be  seen  when 
the  process  of  coagulation  is  observed  through  a  microscope.  After 
a  variable  length  of  time,  more  rapidly  when  the  blood-drop  is  not 
too  small  and  when  the  surface  of  the  glass  is  a  little  uneven,  fine 
filaments  of  fibrin  thus  begin  to  appear,  which  usually  have  for  their 
starting-point  those  bunch-like  conglomerations  of  the  plaques  which 
have  already  been  described.  In  these  bodies  the  pro-enzyme  of  the 
ferment,  the  so-called  prothrombin  of  Alexander  Schmidt,  is  proba- 
bly contained,  and  gives  rise  to  the  ferment  itself  when  the  death 
of  the  cell  occurs.  It  should  be  stated,  however,  that  the  fibrin  fer- 
ment is  not  only  derived  from  the  plaques,  but  may  also  be  formed 
during  decomposition  of  the  remaining  cellular  elements  of  the 
blood,  and,  to  judge  from  recent  observations,  from  protoplasmic 
material  in  general  (see  page  343). 

PHYSICAL  CHARACTERISTICS  OF  THE  BLOOD. 

Color. — The  color  of  normal  blood  is  referable  to  the  presence 
of  a  peculiar  albuminous  substance  in  the  red  cor])uscles  belong- 
ing to  the  class  of  proteids  which  is  termed  haemoglobin.  In 
arterial  blood  this  is  principally  found  in  combination  with  oxy- 
gen as  oxyhsemoglobin,  while  in  venous  blood  a  mixture  of  both 
occurs.  With  a  preponderance  of  oxyhsemoglobin  over  haemoglobin 
the  color  of  the  blood  tends  toward  a  bright  scariet-red.  In  its 
absence  it  assumes  a  dark-bluish  color,  and  we  accordingly  find  all 
gradations  in  shade  between  the  two.  When  venous  blood  is 
exposed  to  the  air  the  haemoglobin  immediately  absorbs  oxygen, 
and  is  transformed  into  oxyhsemoglobin.  This  actually  takes  place 
in  the  alveoli  of  the  lungs,  and  explains  the  difference  in  color 
between  the  blood  of  the  right  and  the  left  heart. 

Under  pathological  conditions  we  may  find  still  other  colors  than 
those  which  have  been  described.  In  coal-gas  poisoning  the  blood 
is  thus  of  a  bright  cherry-red  ;  in  poisoning  with  potassium  chlorate, 


PHYSICAL   CHARACTERISTICS   OF  THE  BLOOD.  331 

anilin,  hydrocyanic  acid,  nitrobenzol,  etc.,  it  is  of  a  brownish-red  or 
a  chocolate  color.  These  changes  are,  as  we  shall  presently  see,  due 
to  certain  chemical  compounds  of  haemoglobin  which  are  not  nor- 
mally found  in  tlie  blood.  In  leukaemia,  in  which  a  most  remark- 
able increase  in  the  leucocytes  may  occur,  the  blood  at  times  pre- 
sents a  milky  appearance.  This  is  not  referable  to  any  change  of 
the  normal  coloring-matter,  however,  but  to  increase  of  the  leucocytes 
as  such. 

The  Odor. — The  odor  of  the  blood  is  characteristic,  but  is  different 
in  different  species  of  animals.  It  can  be  intensified  by  treating  the 
blood  with  a  small  amount  of  fairly  concentrated  sulphuric  acid.  In 
part  it  is  owing  to  the  presence  of  odorous  salts  of  certain  fatty 
acids,  and  to  a  slight  degree  also  to  trimethylamin.  Other  sub- 
stances, however,  are  also  concerned  in  its  production,  but  of  their 
nature  nothing  is  known. 

The  taste  of  blood  is  salty,  but  at  the  same  time  insipid.  It  is 
referable,  no  doubt,  to  the  mineral  constituents  of  the  plasma. 

The  Specific  Gravity. — The  sj)ecific  gravity  of  normal  blood 
seems  to  vary  with  the  amount  of  haemoglobin.  It  is  influenced 
by  the  age  and  sex  of  the  individual,  the  process  of  digestion,  the 
amount  of  exercise  taken,  pregnancy,  etc.  It  is  dependent,  more- 
over, upon  the  bloodvessel  from  which  it  is  drawn,  and  differs 
somewhat  in  different  animals.  Generally  speaking,  it  varies  in 
healthy  adults  between  1.058  and  1.062.  It  is  higher,  as  a  rule,  in 
men  (1.059)  than  in  women  (1.065)  and  in  children  (1.050-1.052). 
Under  pathological  conditions  the  variations  are  much  greater.  It 
may  thus  fall  to  1.025  and  rise  to  1.068.  It  is  to  be  noted,  more- 
over, that  the  specific  gravity  does  not  necessarily  vary  with  the 
amount  of  haemoglobin,  and  in  nephritis,  various  circulatory  dis- 
turbances, in  leukaemia,  and  in  the  anaemias  following  profuse  hem- 
orrhages or  inanition,  care  should  be  had  not  to  draw  inferences 
as  to  the  amount  of  blood  coloring-matter  from  a  determination  of 
the  specific  gravity. 

Determination  of  the  Specific  Gravity. — Hammerschlag's 
Method. — A  cylinder  measuring  about  10  cm.  in  height  is  partly 
filled  with  a  mixture  of  benzol  (sp.  gr.  0.889)  and  chloroform 
(sp.  gr.  1.526),  so  that  the  specific  gravity  lies  between  1.050 
and  i.060.  Into  this  solution  a  drop  of  blood  is  allowed  to 
fall  directly  from  the  finger,  care  being  taken  that  it  does  not 
come  in  contact  with  the  walls  of  the  vessel.  The  drop,  moreover, 
should  not  be  too  large,  as  it  will  otherwise  separate  into  several 
droplets,  and  thus  give  rise  to  inaccurate  results.  It  is  then  brought 
to  suspension  in  the  middle  of  the  fluid,  by  adding  a  little  chloro- 
form or  ether,  according  to  its  tendency  to  sink  to  the  bottom  or  to 
rise  to  the  surface.  As  soon  as  it  remains  in  the  middle  the  mixture 
is  filtered  through  a  layer  of  linen,  and  its  specific  gravity  deter- 
mined by  means  of  an  accurate  hydrometer,  which  is  graduated  to 
the  fourth   decimal.     The    figure    obtained  represents    the    specific 


332  THE  BLOOD. 

gravity  of  the  blood.  It  is  said  that  no  error  is  incurred  through 
evaporation,  and  the  mixture  may  Ije  kept  indefinitely. 

The  Amount. — The  total  amount  of  blood  which  is  contained  in 
tlie  body  corresponds  in  vertebrate  animals  to  from  one-twelfth  to 
one-fourteenth  of  the  body-weight.  It  is  most  conveniently  deter- 
mined according  to  the  following  method  : 

Method  of  Welcker. — From  the  animal  to  be  investigated  10  to 
30  c.c.  of  blood  are  first  withdrawn  and  carefully  defibrinated 
by  whipping.  This  amount  is  weighed  together  with  the  fibrin  and 
set  aside.  The  animal  is  then  bled  to  death  and  the  blood  defibri- 
nated as  before.  After  removal  of  the  feces,  the  intestinal  contents, 
and  the  gall-bladder,  the  entire  body  is  finely  minced  and  repeatedly 
extracted  with  water ;  the  washings  are  added  to  the  large  mass  of 
blood.  The  total  volume  is  now  ascertained  and  the  color  of  the 
bloody  fluid  compared  with  that  of  the  first  10  to  30  c.c,  by 
diluting  this  portion  with  water  until  the  color  of  both  portions 
is  the  same.  From  the  degree  of  dilution,  the  amount  of  blood 
which  is  present  in  the  larger  volume  of  fluid  can  then  readily  be 
determined. 

CHEMICAL  EXAMINATION  OF  THE  BLOOD. 

Reaction. — The  reaction  of  the  blood,  owing  to  the  presence  of 
monosodium  carbonate  and  disodium  phosphate,  is  slightly  alkaline. 
This  may  be  demonstrated  by  repeatedly  drawing  a  strip  of  neutral 
litmus-paper  thoroughly  moistened  with  a  concentrated  solution  of 
common  salt  through  the  blood,  and  rapidly  washing  off  the 
corpuscles  with  the  same  solution.  In  man  the  degree  of  alkalinity 
under  normal  conditions  corresponds  to  from  300  to  325  milli- 
grammes of  sodium  hydrate  for  every  100  c.c.  of  blood.  These  figures 
were  obtained  with  Lowy's  method  (see  below),  and  are  higher  than 
those  usually  given  in  text-books,  but  probably  are  more  nearly 
correct. 

Owing  to  the  formation  of  certain  acids  the  alkalinity  of  the 
blood  rapidly  diminishes  after  being  shed,  and  for  this  reason  its 
determination  is  a  somewhat  difficult  matter.  Generally  speaking, 
it  is  a  little  lower  in  women  and  children  than  in  men,  and  is 
influenced  to  a  certain  degree  by  the  process  of  digestion,  the 
amount  of  exercise  taken,  etc.  At  the  beginning  of  digestion, 
when  hydrochloric  acid  is  being  secreted  in  large  amounts,  it  is  thus 
increased,  while  later  on,  when  the  hydrochloric  acid  and  peptones 
are  reabsorbed,  it  is  diminished.  On  the  whole,  however,  these 
normal  variations  are  slight.  Greater  deviations  have  been  observed 
under  pathological  conditions,  and  are  especiallv  noted  in  leuksemia, 
pernicious  anjemia,  nepliritis,  and  diabetes  when  accompanied  by 
coma,  in  connection  with  high  fever,  during  the  algid  state  of 
Asiatic  cholera,  etc.  It  is  interesting  to  note,  however,  that 
according  to  v.  Limbeck  these  observations  may  be  referable  to 


CHEMICAL  EXAMINATION  OF  THE  BLOOD.  333 

faulty  technique,  as  with  his  method  (see  below)  no  difference  could 
be  shown  to  exist  between  normal  and  pathological  conditions.  It  is 
conceivable,  of  course,  that  in  disease  the  alkalinity  of  the  blood 
may  diminish  more  rapidly  after  being  shed  than  under  normal  con- 
ditions, and  this  may  account  for  the  different  results  which  have 
been  reached  with  other  methods.  But,  on  the  other  hand,  v.  Lim- 
beck's method  may  likewise  not  be  free  from  error. 

The  tenacity  with  which  the  living  organism  tends  to  maintain 
the  normal  composition  of  the  bodily  fluid  is,  of  course,  well  known, 
but  that  it  is  not  always  able  to  do  so  is  also  an  established  fact. 
Herbivorous  animals  thus  rapidly  die  when  given  large  amounts  of 
mineral  acids,  and  it  may  be  shown  that  the  alkalinity  of  their  blood  is 
then  markedlydiminished.  In  cases  of  poisoning  with  strychnin, 
arsenious  acid,  carbon  monoxide,  and  amyl  nitrite,  moreover,  where  a 
marked  albuminous  decomposition  occurs  and  lactic  acid  appears  in 
the  urine,  the  same  result  is  obtained.  Carnivorous  animals,  on  the 
other  hand,  are  more  resistant  in  this  respect,  and  will  stand  much 
larger  amounts.     The  result,  however,  is  the  same. 

Lowy's  Method. — Five  c.c.  of  blood,  obtained  from  one  of  the 
superficial  veins  of  the  arm,  are  allowed  to  flow  into  a  small  flask, 
which  is  provided  with  a  long  and  partially  graduated  neck,  and 
contains  45  c.c.  of  a  0.25  per  cent,  solution  of  ammonium  oxalate. 
Coagulation  is  thus  prevented,  and  the  blood  made  lake-color — /.  e., 
the  hemoglobin  is  dissolved  from  the  stroma  of  the  red  corpuscles. 
The  mixture  is  then  titrated  with  a  one-twenty-fifth  normal  solution 
of  tartaric  acid,  using  as  an  indicator  lacmoid  paper  which  has  been 
soaked  in  a  concentrated  solution  of  magnesium  sulphate.  The 
number  of  cubic  centimeters  employed  to  neutralize  the  5  c.c.  of 
blood,  multiplied  by  0.0016,  will  then  indicate  the  degree  of  alka- 
linity in  terms  of  sodium  hydrate.  The  percentage  is  obtained  by 
multiplying  the  resulting  figure  by  20. 

V.  Limbeck's  Method.— Ten  c.c.  of  blood  are  allowed  to  flow 
into  200  c.c.  of  boiling  water,  to  which  5  c.c.  of  a  one-tenth  nor- 
mal solution  of  hydrochloric  acid  have  been  added._  The  resulting 
solution,  which  is  clear  and  of  a  brownish  color,  is  now  retitrated 
with  a  one-tenth  normal  solution  of  sodium  hydrate,  using  as  indi- 
cator the  syntonin  precipitate  which  occurs  on  neutralization.  The 
difference  between  the  10  c.c.  of  the  hydrochloric  acid  and  the 
sodium  hydrate  solution  is  multiplied  by' 0.004.  The  result  indi- 
cates the  alkalinity  of  the  5  c.c.  of  blood,  and  to  obtain  the  per- 
centage this  is  multiplied  by  20. 

Dare's  Method. — This  mctliod  is  based  upon  tiie  fact  that  the 
characteristic  spectrum  of  oxyha?moglobin  disappears  at  the  point  of 
exact  neutralization  when  the  blood  is  titrated  M'ith  a  dilute  solution 
of  tartaric  acid. 

The  examination  is  made  with  the  aid  of  a  special  instrument,  the 
hccmoalkalimefcr. 

To  neutralize  the  blood,  a  2"^^  normal  solution  of  tartaric  acid  is 


334  THE  BLOOD. 

used,  which  .slioukl  contain  an  amount  of  alcohol  sufficient  to  prevent 
the  growth  of  bacteria,  but  insufficient  to  j)recipitate  the  albumins 
of  the  blood.  The  reagent  may  be  prepared  by  dissolving  0.075 
gramme  of  tartaric  acid  (Merck's  crystals  ;  guaranteed  reagent)  in  a 
small  amount  of  distilled  water,  adding  20  c.c.  of  alcohol  (93-94  per 
cent.),  and  diluting  to  200  c.c.  with  water. 

For  the  spectroscopic  examination  a  Brow'ning  instrument  will 
suffice.     A  detailed  description  accompanies  the  instrument. 

The  Chemical  Composition  of  the  Blood  as  a  Whole. 

As  the  blood  constitutes  the  most  important  channel  through 
which  the  food-stuffs  reach  the  various  tissues  of  the  body,  and 
through  which  waste  matter  is  carried  away,  we  may  expect  to 
find  here  representatives  of  both  classes  of  substances.  This  is 
actually  the  case  ;  but  as  the  waste  matter  is  rapidly  eliminated, 
representatives  of  this  group  are  normally  present  in  only  very 
small  amounts.  Certain  food-stuffs,  moreover,  which  are  not  im- 
mediately required  by  the  body  in  large  quantities,  such  as  fats  and 
carbohydrates,  are  likewise  present  only  in  traces.  They  are  stored 
in  various  tissues  of  the  body  until  needed,  but  even  then  only 
small  quantities  appear  in  the  blood  at  one  time.  The  only  food- 
stuffs, in  fact,  which  are  always  present  in  the  blood  in  large  quan- 
tities, are  the  albumins.  These,  however,  must  be  sharply  separated 
into  two  classes,  viz.,  into  those  which  are  normally  present  as 
integral  constituents  of  the  cellular  elements  of  the  blood,  and  which, 
of  course,  do  not  represent  food-material,  and  into  the  so-called  cir- 
culating albumins  of  the  plasma.  In  addition  to  these  elements,  we 
also  meet  with  mineral  salts,  a  very  large  amount  of  water,  and 
with  certain  grases. 

A  general  idea  of  the  chemical  composition  of  human  blood 
may  be  had  from  the  following  table,  which  is  calculated  for  1000 
parts  by  weight : 

Eed  corpuscles^ 480.00 

Water 276.90 

Oxvhsemoglobin 193.90 

Stroma  2 9.12 

Plasma 520.00 

Water 477.36 

Albumins 35.88 

Extractives 2.39 

Inorganic  salts 4.36 

From  this  analysis  it  will  be  seen  that  almost  one-half  of  the 
total  weight  of  the  blood  is  referable  to  cellular  elements,  and  that 
in  the  liquid  portion  proper  there  is  not  more  than  8.2  per  cent, 
of  .solids,  of  which  6.9  per  cent,  is  albumins,  and  0.84  per  cent, 
mineral  salts  and  0.46   per   cent,  extractives.     The   predominating 

'  The  white  corpuscles,  because  insignificant  in  amount,  have  been  ignored. 
-This  includes  mineral  salts. 


CHEMICAL   EXAMINATION   OF   THE  BLOOD.  335 

solid  substance  in  the  blood  is  the  oxyhsemoglobin  ;  it  represents 
about  19  per  cent,  of  the  total  weight  of  the  blood,  40  per  cent,  of 
the  weight  of  the  blood-corpuscles,  and  95  per  cent,  of  all  organic 
material  present. 

The  native  albumins,  which  are  found  in  the  circulating  blood, 
are  serum-albumin,  serum-globulin,  and  fibrinogen.  The  extractives 
comprise  traces  of  fats,  soaps  of  the  higher  fatty  acids,  lecithin, 
glucose,  animal  gum,  glycogen,  sarcolactic  acid,  urea,  kreatin,  uric 
acid,  and  possibly  also  minimal  amounts  of  the  xanthin  bases. 
Nucleo-albumins,  albumoses,  and  some  of  the  lower  fatty  acids,  oxy- 
butyric  acid,  acetone,  bilirubin,  melanin,  and  other  less  well-known 
bodies  have  further  been  found  under  pathological  conditions,  but 
are  not  seen  in  liormal  blood. 

The  mineral  constituents  comprise  sodium,  potassium,  calcium, 
magnesium,  and  iron.  With  the  exception  of  the  last  mentioned, 
they  are  present  as  chlorides,  phosphates,  carbonates,  and  to  a  slight 
extent  also  as  fluorides.  Some  of  these  occur  in  the  blood  as  such, 
while  others  form  more  or  less  intimate  combinations  with  the  albu- 
mins. Tiie  iron  largely  occurs  as  an  integral  constituent  of  the 
haemoglobin  molecule,  of  which  it  forms  from  0.39  to  0.47  per  cent. 
Traces  are  also  present  in  certain  leucocytes,  and  notably  those  of 
the  oxyphilic  variety.  In  the  plasma  itself  it  is  at  times  met 
with  in  infinitesimally  small  amounts,  and  is  then  referable  to  the 
destruction  of  leucocytes. 

In  addition  to  these  constituents  of  the  normal  blood,  we  further 
meet  with  certain  gases,  viz.,  oxygen,  carbon  dioxide,  and  nitrogen. 
Of  these,  oxygen  and  carbon  dioxide  occur  partly  in  solution  and 
partly  in  combination  with  htemoglobin,  while  nitrogen  is  found 
only  in  solution.  The  carbon  dioxide,  moreover,  is  in  part  present 
as  a  soluble  bicarbonate,  and  to  a  certain  extent  also  in  combina- 
tion with  the  albumins  of  the  plasma.  These  gases  may  be  ex- 
tracted from  the  blood  in  their  entirety  by  exposure  to  a  vacuum.  As 
the  nitrogen  is  simply  held  in  solution,  its  volume  is  constant,  and 
corresponds  to  2  per  cent,  by  volume  no  matter  whether  the  blood 
is  obtained  from  an  artery  or  a  vein.  The  relative  amount  of  oxy- 
gen and  carbon  dioxide,  on  the  other  hand,  is  subject  to  great  varia- 
tions. From  the  arterial  blood  of  dogs  it  is  thus  possible  to  obtain 
21  per  cent,  of  oxygen  by  volume,  and  38  per  cent,  of  carbon  di- 
oxide, while  venous  blood  contains  as  much  as  46  percent,  of  carbon 
dioxide  and  only  12  per  cent,  of  oxygen. 

As  these  gases  can  be  obtained  by  exposure  to  a  vacuum,  it  follow^s 
that  their  combination  with  oxyhsemoglobin  cannot  be  very  strong ; 
it  is  surprising,  however,  to  note  that  in  this  manner  not  only  that 
portion  of  the  carbon  dioxide  is  obtained  which  is  in  combination 
with  albuminous  material,  but  also  the  carbon  dioxide  of  the  car- 
bonates. This  phenomenon  is  owing  to  the  fact  that  in  conse- 
quence of  the  vacuum  the  red  corpuscles  are  broken  down,  and 
that  the  hjemoglo])in  which  is  thus  set  free  is  then  capable  of  exer- 
cising  its  acid  properties,   and  causes  decomposition    of   the  salts. 


336  THE  BLOOD. 


The  Plasma. 


Tn  order  to  obtain  blood-plasma  it  is  necessary  to  prevent  coagu- 
lation of  the  blood.  This  may  be  accomplished  in  various  ways. 
It  has  thus  been  found  that  following  the  intravenous  injection  of 
certain  albumoses  (peptones),  or  of  an  infusion  of  the  mouth  parts 
of  the  officinal  leech,  as  also  after  ligation  of  the  bloodvessels  of 
the  liver  and  intestines,  the  blood  remains  liquid  after  being 
shed.  On  allowing  it  to  stand  at  a  low  temperature  the  blcjod- 
corpuscles  settle  to  the  bottom,  when  the  supernatant  fluid  may 
be  siphoned  off;  or  the  blood  may  be  centrifugalized  at  once  and 
separation  of  the  cellular  elements  effected  in  this  way.  Blood- 
plasma  that  has  been  obtained  after  the  injection  of  albumoses  is 
termed  albumose-plasraa,  or,  less  coYTectXx,  peptone-plasma,  \n  con- 
tradistinction t(j  salt-plasma,  which  results  when  blood  is  received  in 
a  solution  of  a  neutral  salt,  whereby  coagulation  is  also  prevented. 
To  this  end  it  is  best  to  employ  a  saturated  solution  of  sodium 
sulphate  or  a  10  per  cent,  solution  of  sodium  chloride,  to  which  the 
blood  is  added  in  an  equal  amount.  A  saturated  solution  of  magne- 
sium sulphate  may  likewise  be  used,  in  the  proportion  of  one  part 
for  three  parts  of  blood,  but  is  not  so  satisfactory,  as  it  causes  pre- 
cipitation of  certain  albumins  which  are  essential  to  coagulation. 
After  standing  for  twenty-four  hours  the  plasma  may  be  siphoned 
off,  or  may  be  separated  from  the  corpuscles  at  once  by  centrifugation. 

As  cx)agulation  of  the  blood  is  appai'ently  dependent  upon  the 
presence  of  soluble  calcium  salts,  coagulation  may  also  be  prevented 
by  precipitating  with  ammonium  oxalate  those  which  are  present  in 
the  blood.  To  this  end  the  blood  is  received  in  a  solution  of  am- 
monium oxalate,  such  that  the  quantity  of  the  latter  present  in 
the  mixture  amounts  to  about  0.1  per  cent.  This  constitutes 
oxalate-plasma . 

Of  especial  value  in  these  examinations  is  the  blood  of  the  horse, 
in  wiiich  coagulation  occurs  much  more  slowly  than  in  that  of 
mammals.  If  this  is  available,  it  is  only  necessary  to  receive  it  in 
a  narrow  cylinder  surrounded  with  a  freezing-mixture.  Kept  in 
this  manner  it  will  remain  liquid  for  several  days. 

Separated  from  the  corpuscles,  the  plasma  is  a  clear,  straw-colored, 
slightly  viscid  fluid,  of  alkaline  reaction,  and  a  specific  gravitv 
varying  between  1.026  and  1.029  in  man.  It  is  capable  of  under- 
going coagulation,  like  the  native  blood,  and  is  thus  converted  into 
blood-serum.  Its  general  chemical  composition  has  already  been 
considered.  It  contains  but  8.2  per  cent,  of  solids,  of  which  6.9 
per  cent,  is  represented  by  albumins.  These  are  serum-albumin, 
serum-globulin,  and  fibrinogen.  The  relation  between  these  bodies 
is  subject  to  consideral)le  variations.  In  all  animals,  however,  the 
globulins  predoraiimte,  and  in  some  indeed,  as  in  snakes,  serum- 
albumin  is  apparently  absent.  In  the  horse  the  globulins  con- 
stitute about  64.6  per  cent,  of  the  total  amount  of  albumins.     In 


CHEMICAL   EXAMINATION  OF  THE  BLOOD.  337 

1000  parts  by  weight  Hnramarsten  thus  found  38.4  parts  of  serum- 
globulin,  6.5  parts  of  fibrinogen,  and  24.6  parts  of  serum-albumin. 
Of  these  albumins,  fibrinogen  is  of  especial  interest,  as  it  represents 
the  mother-substance  of  fibrin,  and  is  thus  intimately  connected  with 
the  process  of  coagulation. 

Fibrinogen. — Isolation. — Fibrinogen  is  most  conveniently  ob- 
tained from  the  plasma  by  half-saturation  with  sodium  chloride  — 
i.  e.,  by  treating  one  volume  of  the  plasma  with  an  equal  volume  of 
a  saturated  solution  of  common  salt.  The  resulting  precipitate  of 
fibrinogen  is  filtered  off,  washed  with  a  half-saturated  solution  of 
sodium  chloride,  and  dissolved  in  an  8  per  cent,  solution  of  the  salt. 
To  further  purify  the  substance,  this  solution  isreprecipitated,  redis- 
solved,  and  the  process  repeated  twice.  The  final  precipitate  is 
pressed  between  filter-paper  and  suspended  in  water,  in  which  it 
readily  dissolves  owing  to  the  small  amount  of  salt  that  still  remains. 
This  may  be  removed  by  dialysis.  The  purified  substance,  in  a  moist 
state,  appears  in  the  form  of  white  flocculi,  which  readily  coalesce  to 
form  a  tough  elastic  mass. 

The  isolation  of  the  fibrinogen  must  be  performed  rapidly,  as  pro- 
longed exposure  to  the  half-saturated  salt  solution  tends  to  render 
the  substance  insoluble. 

Properties. — Fibrinogen  belongs  to  the  class  of  globulins.  It  is 
insoluble  in  distilled  water,  but  soluble  in  dilute  solutions  of  the 
neutral  salts.  From  these  solutions  it  may  be  precipitated  by  dial- 
ysis, by  increasing  the  amount  of  the  salt,  and  by  passing  a  stream  of 
carbon  dioxide  through  the  solution.  When  kept  under  water  for  a 
comparatively  short  time  it  is  rendered  insoluble.  When  heated  to 
56°  C.  coagulation  occurs,  but  it  appears  that  the  fibrinogen  is  at 
the  same  time  decomposed  into  two  other  globulins,  one  of  which 
coagulates  at  the  temperature  just  mentioned,  while  the  other 
remains  in  solution  until  the  temperature  reaches  65°  C  Of  the 
nature  of  these  two  substances,  however,  we  know  but  little ;  it  is 
possible  that  one  is  the  so-called  fibrinoglobulin,  which,  as  we  shall 
see  later,  is  formed  during  coagulation  of  the  blood.  Fibrinogen 
turns  the  plane  of  polarized  light  to  the  left  ;  its  rotation  for  the 
yellow  D  line  corresponds  to  — 52.5  degrees.  It  consists  of  carbon, 
hydrogen,  nitrogen,  sulphur,  and  oxygen,  in  the  proportion  of  52.93, 
6.9,  16.6,  1.25,  and  22.26,  respectively.  Its  most  characteristic 
property  is  its  tendency  to  the  formation  of  fibrin,  and  upon  this  its 
specific  test  and  quantitative  estimation  are  based.  This  transforma- 
tion will  be  considered  in  detail  later  (see  Coagulation). 

In  addition  to  the  blood-plasma,  fibrinogen  has  been  found  in  the 
chyle,  the  lymph,  and  various  exudates  and  transudates. 

Serum-globulin. — On  half-saturation  of  the  blood-plasma  with 
ammonium  sulphate,  or  on  complete  saturation  with  magnesium 
sul])hate,  an  albuminous  precipitate  is  obtained,  which  was  formerly 
regarded  as  a  unity,  viz.,  as  serum-globulin  {nwe  paraglobulin, 
serum-casein,  Schmidt's  fibrinoplastic  substance).     Later  researches 

22 


338  THE  BLOOD. 

have  shown  that  this  consists  of  several  distinct  bodies,  and  for  this 
reason  it  would  be  better  to  discard  the  term  serum-globulin  as  sucii, 
and  to  speak  of  a  serum-globuliu  fraction.  The  most  notable  com- 
ponents of  this  fraction  are  euglobudn  and  paeudoglobidia.  Tlie 
former  is  precipitated  in  the  presence  of  28-34  per  cent,  of  am- 
monium sulphate,  while  the  latter  is  thrown  down  with  36-44  per 
cent.  These  two  fractions  further  differ  from  each  other  in  the 
behavior  of  their  saline  solutions  on  dialysis.  The  euglobulin  is 
thus  thrown  down  while  the  pseudoglobulin  remains  in  solution. 

Fuld  and  Spiro  have  shown  that  the  coagulating  effect  which  blood 
produces  on  milk  is  referable  to  the  euglobulin  fraction,  while  the 
anti-  action  is  a  property  of  the  pseudoglobulin.  Pick  has  demon- 
strated still  a  further  difference  ;  in  horses  immunized  to  diphtheria 
the  antitoxic  properties  of  the  serum  are  connected  with  the  pseudo- 
glol)uliu,  while  the  other  fraction  is  inert. 

As  these  two  fractions  in  their  general  properties  are  otherwise 
apparently  identical,  it  has  been  suggested  that  tiie  difference  in  their 
behavior  to  ammonium  sulphate  and  on  dialysis,  etc.,  might  possi- 
bly be  due  to  a  third  factor.  Morner  tlms  thinks  that  contamina- 
tion of  the  globulin  precipitate  by  soaps,  fitty  acids,  lecithins,  etc., 
is  the  determining  factor  in  this  respect.  The  majority  of  physio- 
logical chemists,  inclusive  of  Hammarsten,  who  has  been  especially 
active  in  this  field,  however,  incline  to  the  view  that  serum-globulin 
represents  a  mixture  of  at  least  two  or  more  bodies. 

Freund  and  Joachim  have  recently  pointed  out  that  from  both 
euglobulin  and  pseudoglobulin  they  could  obtain  one  fraction  which 
was  precipitated  on  dialysis  and  a  second  one  which  remained  in 
solution.  The  water-insoluble  portion  of  each  fraction  they  desig- 
nate as  para-euglobulin  and  para-pseudoglobulin  respectively,  while 
the  terms  euglobulin  and  pseudoglobulin  are  retained  for  the  water- 
soluble  portion  of  each  fraction. 

Serum-globulin,  using  the  term  in  the  older  sense  of  the  word,  is 
found  not  only  in  the  plasma  of  the  bhiod,  but  also  in  the  lymph,  in 
various  exudates  and  transudates,  in  the  serum,  in  the  white  and  red 
corpuscles  of  the  blood,  and  in  traces  at  least  in  all  the  cellular  ele- 
ments of  the  body  (cell-globulins)  In  the  urine  serum-globulin 
invariably  accompanies  serum-albumin. 

Properties. — As  a  class  the  serum-globulins  represent  a  snowv- 
white,  finely  flocculent  material^  which  is  not  tough  and  elastic  like 
fibrinogen.  They  have  not  been  obtained  in  crystalline  form.  In 
water  the  euglobulins  are  practically  insoluble,  while  the  pseudo- 
globulins  will  dissolve.  The  insolubility  in  water  was  formerly 
regarded  as  one  of  tlie  characteristic  features  of  the  globulins.  In 
dilute  saline  solution  they  are  all  soluble  with  comparative  readi- 
ness,  l)ut  on  standing  the  substance  gradually  l)ecomes  insolul)le. 

From  their  solutions  the  globulins  are  precipitated  by  half-satura- 
tion with  ammonium  sulphate,  or  by  complete  saturation  with 
magnesium  sulphate.     Sodium  chloride  causes  only  an  incomplete 


CHEMICAL  EXAMINATION  OF  THE  BLOOD.  339 

separation  when  added  to  saturation,  and  with  half-saturation  no 
effect  at  all  is  produced.  It  is  thus  an  easy  matter  to  isolate 
serum-glol)ulin,  even  though  fibrinogen  be  present  at  the  same  time. 
An  incomplete  precipitation  occurs  when  its  neutral  or  feebly  acid 
solutions  (using  acetic  acid)  are  diluted  from  10  to  2U  times  with  dis- 
tilled water,  or  on  passing  a  stream  of  carbon  dioxide  through  such 
dihite  solutions.  In  the  presence  of  5  to  10  ])er  cent,  of  sodium 
chloride  the  globulin  fraction  coagulates  at  75°  C. 

Elementary  analysis  of  the  entire  fraction,  as  maybe  expected, 
has  not  given  rise  to  constant  results.  An  analysis  by  Hammarsten 
gave  the  following  figures  :  C  =  52.71  ;  H  =  7.01  ;  N  =  15.85  ; 
8=  1.11  ;0  =  23.32. 

Isolation. — Serum-globulin  is  most  conveniently  obtained  from 
blood-serum  by  half-saturation  with  ammonium  sulphate — i.  e.,  by 
treating  a  given  volume  of  tlie  serum  with  the  same  amount  of  a 
saturated  solution  of  the  salt.  Saturation  w^ith  magnesium  sulphate 
in  substance  may  also  be  em]>loyed.  In  either  case  the  precipitated 
serum-globulin  is  filtered  off,  washed  with  the  corresponding  salt 
solution,  dried  at  115°  C,  then  washed  with  boiling  water  to  remove 
the  remaining  salts,  extracted  with  alcohol,  then  with  ether,  and 
finally  dried  and  weighed.  In  any  case  the  original  solution  should 
be  nearly  neutral  in  reaction. 

Serum-albumin. — Serum-albumin  is  found  in  the  plasma,  the 
serum,  the  lymph,  in  exudates  and  transudates,  and  under  certain 
pathological  conditions  also  in  the  urine,  where  it  usually  occurs  in 
association  with  serum-globulin. 

In  the  dry  state  serum-albumin  is  a  transparent,  gum-like,  brittle, 
hygroscopic  mass,  or  a  white  powder,  which  can  be  heated  to  100°  C. 
witiiout  undergoing  decomposition.  Solutions  of  tiie  pure  substance 
in  distilled  water  coagulate  at  50°  C,  while  in  the  presence  of  salts 
a  higher  temperature  is  necessary.  This  varies  with  the  amount  of 
salt  present,  as  also  with  the  concentration  of  the  albumin.  A  1 
to  2  per  cent,  solution  containing  5  per  cent,  of  sodium  chloride 
coagulates  between  75°  and  90°  C  From  its  salt  solutions  serum- 
albumin  may  be  obtained  in  crystalline  form.  Its  specific  rotation 
in  distilled  water  varies  between  62.6°  and  64.6°. 

One  of  tlie  most  remarkable  properties  of  serum-albumin  is  its 
pronounced  tendency  to  form  a  sulphate.  It  is  manifestly  capable 
of  abstracting  sulphuric  acid  (not  sulphates)  from  the  sulphates 
used  in  its  isolation,  and  this  sulphur  cannot  be  removed  by  wash- 
ing with  water  (Morner). 

According  to  Halliburton,  the  serum-albumin  of  mammalian 
blood-serum  is  not  a  single  substance,  but  consists  of  three  distinct 
albumins,  which  he  terms  «-,  /9-,  and  ^'-serum-albumin.  They  are 
said  to  coagulate  at  73°  C,  77°  C,  and  84°  C,  respectively.  In 
cold-blooded  animals,  «-serum-albumin  only  is  said  to  occur. 

More  recently  Op])enheimer  iias  shown  that  by  salting  with 
ammonium  sulphate  serum-albumin  can  be  divided  into  two  frac- 


340  THE  BLOOD. 

tioiis,  one  of  which   is   thrown   down   at   6t)f  per  cent,  saturation, 
while  the  second  fraction  is  precipitated  on  82  per  cent,  saturation. 

Isolation. — Serum-albumin  is  most  conveniently  obtained  from 
])lood-serum  after  removal  of  the  serum-globulin  by  saturation  with 
magnesium  sulphate  at  a  temperature  of  30°  C.  The  filtrate  is 
saturated  with  sodium  sulphate  or  ammonium  sulphate  at  40°  C,  or 
treated  with  acetic  acid,  so  that  the  solution  contains  about*  1  per 
cent.  In  either  case  the  precipitated  serum-albumin  is  filtered  ofl^', 
pressed  between  layers  of  filter-paper,  dissolved  in  water  (the  reac- 
tion should  be  neutral),  and  separated  from  the  remaining  salt  by 
dialysis.  From  its  aqueous  solutions  it  is  finally  obtained  by 
evaporation  at  a  low  temperature  or  by  precipitation  with  alcohol, 
which  must  be  rapidly  removed,  however,  as  otherwise  it  will  cause 
coagulation  of  the  albumin. 

Separation  of  the  Albumins  of  the  Blood-plasma  from  Each 
Other. — To  isolate  the  fibrinogen,  the  plasma  is  treated  with  an 
equal  volume  of  a  saturated  solution  of  sodium  chloride.  The  result- 
ing precipitate  is  filtered  off  and  purified  as  described.  The  filtrate 
contains  serum-globulin  and  serum-albumin.  The  globulin  is  pre- 
cipitated bv  saturation  with  magnesium  sulphate,  filtered  off,  and 
likewise  purified.  The  filtrate  contains  only  the  serum-albumin, 
which  may  be  obtained,  as  just  described,  by  saturation  with  sodium 
or  ammonium  sulphate. 

Quantitative  Estimation  of  the  Total  Albumin  of  the  Plasma. 
— The  albumins  are  most  conveniently  estimated  by  treating  a  care- 
fully measured  and  weighed  amount  of  the  plasma,  after  neutraliza- 
tion with  acetic  acid.  Math  five  times  its  volume  of  alcohol.  After 
standing  for  twenty-four  hours  the  solution  is  boiled  for  several 
minutes,  and  the  resulting  precipitate  collected  on  a  weighed  filter, 
washed  with  hot  alcohol,  then  with  ether,  dried  at  115°  C,  weighed, 
and  incinerated.  The  weight  of  the  ash  is  deducted  from  the  weight 
of  the  precipitate. 

More  accurate  is  the  following  method :  a  carefully  measured 
and  weighed  amount  of  the  plasma  is  treated  with  one-half  its  volume 
of  a  saturated  solution  of  sodium  chloride  and  a  slight  excess  of 
tannic  acid.  In  the  resulting  precipitate  the  nitrogen  is  then  esti- 
mated according  to  Kjeldahl's  method.  When  multiplied  by  6.37 
the  corresponding  amount  of  albumin  is  obtained. 

To  remove  all  albumins  from  the  blood,  Cavazzani's  method  may 
be  employed.  To  this  end,  20-30  c.c.  of  blood  are  added  to  200  c.c. 
of  distilled  water,  and  treated  with  five  or  six  drops  of  a  solutioa 
consisting  of  ten  parts  of  acetic  acid  (sp.  gr.  1.040)  and  one  part  of 
lactic  acid.  The  mixture  is  boiled  for  about  ten  minutes,  filtered, 
and  the  i)recipitate  washed  separately  with  hot  water,  and  finally 
pressed  in  a  piece  of  muslin.  The  filtrate  and  washings,  which  are 
practically  colorless,  are  then  concentrated  to  a  small  volume.  Any 
traces  of  albumin  which  may  still  be  present  thus  separate  out  and 
are  filtered  off.     If  too  much  of  the  acid  solution  has  been  added,  the 


CHEMICAL   EXAMINATION  OF  THE  BLOOD.  341 

mixture  may  not  clear  on  boiling.  In  that  event  a  few  crystals  of 
sodium  carbonate  are  added,  when  coagulation  promptly  occurs.  On 
the  other  hand,  it  may  at  times  be  necessary  to  add  a  few  drops 
more  of  the  acid  solution. 

The  remaining  constituents  of  the  plasma  are  also  found  in  the 
serum,  and  will  be  considered  in  that  connection. 

The   Serum. 

The  serum  results  from  the  blood-plasma  during  the  process  of 
coagulation.  It  is  most  conveniently  obtained  by  whipping  blood 
immediately  after  being  shed,  whereby  the  greater  portion  of  the 
fibrin  is  removed  and  the  formation  of  large  clots  prevented.  The 
corpuscles  and  smaller  pieces  of  fibrin  are  separated  by  centrifuga- 
tion  or  by  allowing  the  fluid  to  stand  in  tlie  cold  until  sedimen- 
tation has  occurred.  The  serum  is  then  siphoned  off  and  filtered. 
It  thus  appears  as  a  slightly  viscid,  fairly  transparent  fluid  of  a  light 
straw  color,  which  presents  a  feebly  alkaline  reaction  and  a  specific 
gravity  varying  between  1.026  and  1.029  in  man.  In  its  chemical 
composition  scrum  differs  from  plasma  principally  in  the  presence 
of  the  fibrin  ferment  and  in  the  absence  of  fibrinogen.  In  its  place, 
however,  traces  of  two  other  globulins,  which  are  not  present  in  the 
plasma,  are  found.  One  of  these  is  termed  fibrinoglobulin,  and  is 
thought  to  result  during  the  formation  of  fibrin  from  fibrinogen. 
The  other  is  the  so-called  cell-globulin,  and  is  supposedly  referable 
to  the  decomposition  of  leucocytes  during  the  process  of  coagulation. 
The  remaining  constituents  are  qualitatively  the  same  in  both  fluids. 
Slight  quantitative  differences,  however,  exist,  A  portion  of  the 
calcium,  magnesium,  and  phosphoric  acid  is  thus  eliminated 
together  with  the  fibrin,  and  accordingly  lower  values  are  found 
in  the  serum  than  in  the  plasma. 

An  idea  of  the  mineral  constituents  of  the  serum,  and  their 
quantitative  relations,  may  be  had  from  the  accompanying  table  : 

Man. 

Potassium  oxide 0.387 — 0.401  pro  mille. 

Sodium  oxide 4.290  " 

Chlorine      3.565—3.659  " 

Calcium  oxide 0.155  " 

Magnesium  oxide 0.101  " 

From  this  it  will  be  seen  that  sodium  in  the  form  of  the  chloride 
largely  predominates  in  the  serum,  while  potassium  occurs  only  in 
small  amounts.  This  is  exactlv  the  reverse  of  what  is  seen  in  the 
morphological  elements  of  the  body,  of  whicli  potassium  comj^ounds 
are  the  principal  salts  present.  It  is  noteworthy,  moreover,  that  the 
amount  of  sodium  chloride  is  practically  constant  in  the  blood,  no 
matter  whether  large  quantities  are  ingested  or  the  salt  is  given  in  only 
small  amounts.     During  starvation  even,  or  when  the  potassium  salt 


342  THE  BLOOD. 

is  artificially  substituted,  the  amount  present  in  the  blood  remains 
practically  constant.  Apparently  it  occurs  only  in  solution,  and  does 
not  form  an  integral  part  of  the  albuminous  molecule,  as  is  the  case 
with  the  phosphates  of  the  blood.  Of  these,  traces  only  are  present 
in  solution,  while  the  greater  portion  is  more  or  less  intimatelv  com- 
bined with  the  albumins.  As  the  lecithins  which  are  found  in  the 
blood  also  contain  phosphorus,  it  follows  that  these  figures,  which  are 
obtained  by  incinerating  a  given  amount  of  serum  or  plasma  and 
determining  the  phosphoric  acid  in  the  ash,  are  too  high.  In  serum 
which  had  been  freed  from  lecithin  Sertoli  and  ISIroczkowski  found 
amounts  varying  between  0.02  and  0.09  pro  mille,  calculated  as  di- 
sodium  phosphate.  In  the  estimation  of  the  sulphates  we  meet 
with  still  greater  difficulties,  as  the  sulphur  of  the  albumins  is 
included  in  the  determination.  The  amount  which  is  present  in 
solution,  however,  is  certainly  very  small.  The  iron  which  is  at 
times  met  with  in  the  serum  is  unquestionably  derived  from  the 
leucocytes,  and  is  an  accidental  constituent.  The  amount  which 
may  be  obtained  is  always  exceedingly  small. 

Of  other  elements,  traces  of  silicon,  fluorine,  copper,  and  man- 
ganese have  at  times  been  observed. 

The  coloring-matter  of  the  serum  and  plasma  is  supposedly  due 
to  a  substance  belonging  to  the  class  of  lipochromes  or  luteins. 

The  albumins  of  the  serum  which  also  occur  in  the  plasma,  viz., 
serum-albumin  and  serum-globulin,  have  been  considered.  Of  the 
fibrinoglobulin  which  is  formed  during  coagulation  of  the  blood, 
and  which  is  thought  to  result  from  decomposition  of  fibrinogen, 
comparatively  little  is  known.  It  coagulates  at  64°  C,  and  appar- 
ently represents  about  one-third  of  the  fibrinogen  molecule.  Of  the 
so-called  cell-globulin,  still  less  is  known,  and  both  are  found  only 
in  traces. 

The  Coagulation  of  the  Blood. 

The  Fibrin-ferment. — The  coagulation  of  the  blood  is  supposedly 
referable  in  the  first  instance  to  the  decomposition  of  fibrinogen  into 
fibrin  and  fibrinoglobulin.  This  decomposition  is  thought  to  be 
effected  through  the  activity  of  a  special  Jibrin-fenncnf,  the  thrombin 
of  A.  Schmidt,  which  is  supposedly  present  in  the  cellular  elements 
of  the  blood  as  a  pro-enzyme,  Schmidt's  profhrombiti,  and  is  set 
free  after  the  death  of  these  elements  as  a  calcium  compound  of 
the  enzyme.  In  the  circulating  blood  the  ferment  is  manifestly 
not  present,  as  its  solution,  when  injected  into  the  bloodvessels 
of  a  living  animal,  will  cause  almost  instantaneous  death  from 
thrombosis. 

According  to  Pekelharing,  the  coagulating  action  of  the  ferment 
is  the  outcome  of  the  transference  of  the  calcium  of  the  ferment  to 
the  fibrinogen,  which  is  decomposed  with  the  formation  of  insoluble 
calcium  fibrin.  As  a  result  the  ferment  is  reconverted  into  the  pro- 
enzyme, and  as  such  immediately  combines  with  a  new  ptr  "tion  of 


CHEMICAL   EXAMINATION  OF  THE  BLOOD.  343 

calcium,  which  is  again  transferred  to  another  portion  of  fibrinogen, 
and  so  on. 

Another  view  regarding  the  coagulation  of  the  blood  is  that  ex- 
pressed by  Lilienfeld.  While  not  denying  the  existence  of  a  fibrin- 
ferment,  he  regards  this  as  a  decomposition-product  which  is  formed 
during  the  pi'ocess  of  coagulation,  and  which  accordingly  cannot  be 
concerned  in  the  process  itself.  He  assumes  that  coagulation  of  the 
blood  is  normally  effected  by  the  acid  radicle  of  nucleohistou,  which 
is  present  in  the  nuclei  of  leucocytes  in  large  amount. 

The  leuconuclein,  however,  does  not  cause  coagulation  of  the 
blood  at  once,  but  first  effects  the  decomposition  of  fibrinogen  with 
the  formation  of  thrombosin  and  an  albumose-like  body,  which  may 
be  a  derivative  of  Harnmarsten's  fibrinoglobulin.  The  thrombosin 
is  soluble  in  dilute  alkalies.  Upon  the  addition  of  a  soluble  calcium 
salt,  however,  such  solutions  coagulate,  and  the  thrombosin  is  thus 
transformed  into  the  insoluble  fibrin.  Fibrin,  according  to  this 
theory,  is  thus  a  calcium  compound  of  thrombosin. 

The  active  principle  of  the  leuconuclein  is  unquestionably  its 
nucleinic  acid  radicle,  and,  as  a  matter  of  fact,  the  same  changes  can 
be  brought  about  by  using  this  directly.  This  explains  the  ol)serva- 
tion  which  has  been  repeatedly  made,  that  the  coagulation  of  albu- 
minous fluids  which  are  not  spontaneously  coagulable  can  also  be 
effected  by  the  addition  of  almost  any  cellular  elements,  such  as 
yeast  cells,  various  bacteria  and  moulds,  spermatozoa,  protozoa, 
etc. — i.  e.,  bodies  which  all  contain  fairly  large  amounts  of  nucleins. 
It  is  to  be  noted,  moreover,  that  the  intensity  of  action  of  these  vari- 
ous substances  is  intimately  dependent  upon  the  amount  of  nuclein 
present,  and  we  accordingly  find  that  of  all  the  cellular  elements  of 
the  body  the  spermatozoa  are  the  most  active.  While  this  portion 
of  Lilienfeld's  theory  is  practically  unassailable,  some  doubt  has 
been  raised  as  to  the  existence  of  his  soluble  thrombosin. 
Hamraarsten  thus  states  that  the  thrombosin  is  no  decomposition- 
product  of  fibrinogen,  but  fibrinogen  itself  which  has  been  precipi- 
tated with  nucleinic  acid,  a  view  which  has  been  proved  to  be  cor- 
rect by  Cramer.  He  has  shown,  moreover,  that  the  formation  of 
fibrin  can  take  place  in  the  absence  of  calcium  salts,  providing  that 
a  sufficient  amount  of  the  fibrin-ferment  is  present.  If  we  are  to 
accept  Lilienfeld's  theory  then,  we  may  imagine  that  through  the 
influence  of  the  soluble  calcium  salt  the  nucleohiston  is  decomposed, 
and  that  the  leuconuclein  is  thus  enabled  to  exercise  its  special  ac- 
tivity. 

According  to  Pekelharing,  the  fibrin-ferment  is  identical  with  a 
nucleoproteid  which  he  found  in  the  serum,  and  which  in  turn  may 
be  derived  from  the  leucocytes  where  it  has  also  been  demonstrated. 
Its  coagulating  properties,  however,  are  here  also  dependent  upon 
the  presence  of  a  calcium  salt.  As  in  the  case  of  the  nucleohiston 
(the  coagulating  properties  of  which  he  admits),  the  amount  of  cal- 
cium, according  to  Huiskamp,  is  in  both  cases  of  primary  importance. 


344  THE  BLOOD. 

If  no  calcium  salt  is  present,  no  coagulation  occurs  even  though  the 
nucleohiston  and  the  nucleoproteid  be  present.  In  the  presence  of 
a  very  small  amount  of  calcium  they  are  in  part  precipitated,  and 
only  bring  about  a  slow  and  incomplete  coagulation  ;  in  the  presence 
of  somewhat  more,  viz.,  0.1-0.3  per  cent,  of  calcium  chloride,  they 
are  quite  completely  precipitated  and  complete  coagulation  rapidly 
occurs.  In  the  presence  of  0.5-0.6  per  cent,  of  calcium  chloride 
nucleohiston  just  remains  precipitated,  or  perhaps  just  begins  to 
dissolve,  while  the  nucleoproteid  is  in  great  part  dissolved  ;  coagula- 
tion now  again  occurs  slowly.  In  the  presence  of  0.8  per  cent, 
nucleohiston  is  all  dissolved  and  the  nucleoproteid  to  a  great  extent, 
and  coagulation  does  not  occur.  According  to  Huiskamp,  the 
nucleoproteid  may  be  regarded  as  the  prothrombin  and  the  calcium 
compound  as  the  thrombin.  This  view  is  possibly  too  extreme,  and 
just  doubts  have  been  raised  as  to  the  identity  of  Huiskamp's  nucleo- 
proteid with  the  iibrin-ferment. 

These  conflicting  opinions  may  be  adjusted  if  we  assume  the  pos- 
sible existence  of  more  than  one  class  of  substances  which  are  cap- 
able of  causing  coagulation  of  the  blood,  viz.,  of  actual  ferments 
on  the  one  hand,  and  pseudoferments  on  the  other. 

Of  special  interest  in  this  connection  is  the  fact  that  the  true  fer- 
ment (according  to  Schmidt)  cannot  be  kept  in  solution  and  retain 
its  activity.  It  becomes  inactive,  but  can  under  certain  conditions 
be  activated  again  by  the  addition  of  an  alkali,  to  become  inactive 
again  on  neutralization,  etc.  Fuld  suggests  that  this  change  of  phase 
may  be  the  deciding  factor  which  enables  the  thrombin  to  cause 
coagulation. 

Isolation. — The  isolation  of  the  fibrin-ferment  (in  the  sense  of  A. 
Schmidt)  is  most  conveniently  accomplished  in  the  following  man- 
ner :  Taking  the  serum  of  the  ox,  the  globulins  are  first  precipitated 
by  saturation  with  magnesium  sulphate.  The  filtrate  is  then  diluted 
M'ith  water,  and  treated  while  stirring  with  a  very  dilute  solution  of 
sodium  hydrate  until  an  abundant  and  flocculent  precipitation  cf 
magnesium  hydroxide  has  been  brought  about.  This  preci])itate, 
which  contains  a  large  proportion  of  the  ferment,  is  washed  with 
water,  pressed  between  filter-paper,  and  dissolved  in  water  liy  neu- 
tralizing the  solution  with  diluted  acetic  acid.  The  salts  are  then 
removed  by  dialysis,  when  the  ferment  can  be  precipitated  l)y  a 
suitable  addition  of  acetic  acid. 

Properties. — Of  tlie  nature  of  the  product  which  can  thus  be  ob- 
tained little  is  known.  On  digestion  with  pepsin  it  yields  a  nuclein, 
and  is  accordingly  regarded  as  a  nucleoproteid  (Pekelharing).  As 
is  the  case  with  all  ferments,  its  solutions  are  rendered  inactive  by 
exposure  to  a  moderately  high  temperature  (70°-75°  C),  while  the 
various  antiseptic  substances  which  do  not  interfere  with  the  activity 
of  other  ferments  are  likewise  without  effect  upon  the  fibrin-ferment. 
Its  specific  activity  is  manifested  when  brouglit  into  contact  with 
fibrinogen,  which  is  apparently  decomposed  by  hydrolysis  into  fibrin 


CHEMICAL   EXAMINATION  OF  THE  BLOOD.  345 

and  fibrinoglobulin.  To  effect  this  change,  however,  the  presence 
ot'a  neutral  salt  and  of  a  soluble  calcium  salt  is  essential.  In  their 
absence  coagulation  does  not  take  place. 

To  ted  for  the  fibrin-ferment,  Arthus  has  suggested  the  use  of 
fluoride-plasma  of  dogs.  In  such  plasma,  as  also  in  the  correspond- 
ing product  of  horses  (Fuld),  coagulation  cannot  be  brought  about 
without  the  ferment;  neither  the  addition  of  a  calcium  salt  nor  at- 
tempts at  activation  by  treating  a  portion  with  alkali  and  adding  it 
to  the  rest  will  succeed.  If  then  coagulation  does  occur  after  adding 
a  solution  to  be  tested,  we  may  infer  that  the  flbrin-ferment  was 
present  in  the  latter. 

Fibrin. — Fibrin  is  formed  during  the  spontaneous  coagulation  of 
all  albuminous  solutions  which  contain  fibrinogen  and  cellular  ele- 
ments that  can  give  rise  to  the  fibrin-ferment.  It  is  most  conveni- 
ently obtained  by  whipping  freshly  shed  blood  with  a  suitable  instru- 
ment, when  the  fibrin  is  deposited  as  an  elastic,  stringy  material, 
which  may  be  freed  from  adhering  corpuscles  by  tliorough  washing 
and  kneading  in  running  water.  Such  fibrin,  however,  is  still  con- 
taminated with  serum-globulin  and  certain  phosphorus-containing 
substances  which  have  resulted  from  the  decomposition  of  leucocytes. 
The  serum-globulin  may  be  removed  by  separate  washing  and  knead- 
ing in  a  5  per  cent,  solution  of  common  salt ;  but  the  other  products, 
as  well  as  the  remains  of  the  corpuscles  of  the  blood,  can  scarcely  be 
removed.  To  obtain  pure  fibrin,  therefore,  it  is  necessary  to  start 
with  filtered  plasma  or  with  filtered  transudates,  which  are  beaten 
with  a  piece  of  whalebone,  after  adding  a  little  serum,  if  the  fluid 
is  not  spontaneously  coagulable.  The  resulting  material  is  washed 
with  water,  then  with  a  5  per  cent,  solution  of  sodium  chloride,  and 
finally  extracted  with  alcohol  and  ether. 

The  fibrin  then  appears  as  a  white  stringy  substance,  which  is 
somewhat  elastic,  but  is  easily  rendered  brittle  on  contact  \\\\\\ 
alcohol  or  on  warming  the  substance  in  water  to  a  temperature  of 
75°  C.  It  is  closely  related  to  the  coagulated  albumins,  and  ac- 
cordingly is  soluble  only  with  difficulty.  It  is  questionable,  more- 
over, whether  solution  of  the  substance  can  be  accomplished  without 
causing  its  decomposition.  If  fibrin  is  thus  placed  in  a  5  to  10  per 
cent,  solution  of  sodium  chloride,  or  a  6  per  cent,  solution  of  sodium 
nitrate,  and  kept  at  a  temperature  of  40°  C,  it  first  swells  up  and 
gradually  disappears  as  such.  In  its  place  two  globulins  or  related 
substances  are  then  said  to  be  found.  This  transformation,  accord- 
ing to  some  observers,  is  referable  to  adherent  bacterial  enzymes. 
In  dilute  alkalies  and  acids  it  likewise  dissolves.  Stronger  acids, 
as  also  the  proteolytic  ferments,  dissolve  the  fibrin,  but  at  the 
same  time  cause  its  transformation  into  acid  albumin  and  albumoses. 
In  Avater,  alcohol,  and  ether  it  is  entirely  insoluble. 

The  elemcntarv  analysis  of  fil)rin  gives  52.68  parts  of  carbon, 
6.83  of  hydrogen,  16.41  of  nitrogen,  1.1  of  sulphur,  and  22.48 
of  oxygen.     It  is  to  be  noted,   further,    that  in  addition  to    these 


346  TEE  BLOOD. 

elements  calcium  is  constantly  present,  and,  as  has  been  seen,  its 
formation  is  largely  dependent  upon  the  presence  of  a  soluble 
calcium  salt. 

The  amctunt  of  fibrin  which  may  be  obtained  from  the  blood, 
notwithstanding  ils  bulk,  does  not  exceed  0.1-0.4  per  cent. 

Estimation. — In  order  to  determine  the  amount  of  librin  in  a 
given  volume  of  blood,  from  30  to  40  c.c.  are  placed  in  a  previously 
weighed  beaker,  wdiich  is  closed  with  an  India-rubber  cap.  Through 
the  centre  of  this  passes  a  piece  of  whalebone  that  is  firmly  fixed 
and  provided  with  a  rudder-like  end,  "which  dips  into  the  blood. 
This  is  now  dcfibriuated  by  beating  with  the  whalebone  paddle, 
when  the  beaker  is  again  weighed  with  its  contents.  The  differ- 
ence, as  compared  with  the  first  weight,  indicates  the  weight  of  the 
blood.  The  beaker  is  then  filled  with  water  and  the  mixture  again 
beaten.  The  fibrin  is  allowed  to  settle,  and  after  being  washed  by 
decantation  with  normal  salt  solution  it  is  collected  on  a  filter  of 
known  weight.  On  this  it  is  further  washed  with  normal  salt  solu- 
tion until  free  from  coloring-matter  ;  it  is  then  extracted  with  boiling 
alcohol,  then  with  ether,  and  finally  dried  at  115°  C  and  weighed. 

The  question,  why  the  blood  does  not  coagulate  within  the  vessels 
of  the  body  where  fibrinogenic  material  is  available,  and  where  leuco- 
cytes no  doubt  undergo  degeneration,  has  been  variously  answered. 
On  the  one  hand,  it  is  stated  that  the  integrity  of  the  endothelial 
lining  is  here  of  prime  importance,  and  that  coagulation  will  occur 
whenever  this  is  impaired.  As  a  matter  of  fact,  we  find  that  coagu- 
lation takes  place  after  ligation  of  an  artery,  and  that  the  coagulum 
invariably  extends  as  far  as  the  next  collateral  vessel.  That  the 
nutrition  of  the  intima  is  here  seriously  interfered  with  cannot  be 
doubted.  Similarly  we  find  a  more  or  less  extensive  thrombosis 
in  atheromatous  vessels,  not  to  speak  of  the  process  of  clotting  in 
association  with  wounds.  In  such  cases  it  appears  that  owing  to  the 
lesion  of  the  endothelial  coat  an  aggregation  of  leucocytes  occurs 
in  the  affected  parts,  which  in  turn  results  in  the  death  and  disso- 
lution of  many  of  the  cells  at  these  places.  Contact  with  a  foreign 
substance,  and  as  such  diseased  or  dying  endothelial  cells  must  be 
viewed,  in  some  manner  brings  about  the  early  dissolution  of  the 
leucocytes,  and  we  find  accordingly  that  on  introducing  a  silk  thread 
into  the  bloodvessel  of  a  living  animal  coagulation  takes  place 
around  the  foreign  body.  Similarly,  it  may  be  observed  that  when 
blood  is  received  in  a  vessel,  the  walls  of  which  have  been  carefully 
lubricated  with  vaselin,  coagulation  is  greatly  delayed,  but  may  be 
brought  about  at  once  on  introducing  bits  of  foreign  material,  such 
as  dust  or  ashes  and  the  like. 

AYhile  we  have  thus  seen  that  coagulation  will  occur  within  the 
living  body  whenever  foreign  material  is  present,  w'e  have  not  as  yet 
offered  an  explanation  for  the  non-occurrence  of  coagulation  wdien 
such  influences  are  not  at  work.  That  leucocytes  are  constantly 
broken  down  in  the  living  organism  cannot   be  questioned.     It  is 


CHEMICAL  EXAMINATION  OF  THE  BLOOD.  347 

possible,  however,  that  the  amount  of  nucleohiston,  or  fihrin-fcrnient, 
as  the  case  may  be,  which  is  thus  formed,  is  too  small  at  any  one 
time  to  exert  its  special  activity.  On  the  other  hand,  it  is  conceiv- 
able that  such  decomposition  may  take  place  within  certain  organs 
of  the  body,  such  as  the  spleen  and  the  liver,  and  that  the  nucle- 
histon,  which  is  here  set  free,  is  again  utilized  in  the  construction  of 
new  cells.  The  nucleohiston,  moreover,  contains  another  radicle, 
the  albumose-like  histon,  which  has  exactly  the  opposite  effect  upon 
coagulation.  For  whereas  the  injection  of  leuconuclein  into  the  cir- 
culation of  living  animals  rapidly  brings  about  the  death  of  the 
animal  from  thrombosis,  histon  injections  render  the  blood  refractory 
to  coagulation.  Such  plasma  is  termed  histon-plasma,  and  it  is  to 
be  noted  that  this  coagulation  can  later  be  brought  about  only  through 
the  addition  of  the  leuconuclein  or  nucleinic  acid.  It  is  thus  possi- 
ble that  during  the  dissolution  of  the  leucocytes  in  the  living  animal 
the  leuconuclein  is  in  some  manner  prevented  from  exercising  its 
special  activity,  while  the  histon  further  prevents  coagulation  in 
itself.  The  primary  factor,  however,  upon  which  the  occurrence  or 
non-occurrence  of  coagulation  in  the  living  body  probably  depends, 
is  the  extent  to  which  the  leucocytes  are  undergoing  decomposition. 

Rapidity  of  Coagulation. — The  rapidity  with  which  coagu- 
lation of  the  blood  occurs  after  being  shed  varies  with  different 
animals,  with  the  districts  from  which  the  blood  is  taken,  etc. 
In  birds  it  thus  occurs  after  one  and  a  half  minutes ;  in  man 
after  from  three  to  four  minutes ;  while  in  cold-blooded  animals 
it  begins  only  after  a  quarter  of  an  hour.  In  the  horse,  in  which 
coagulation  is  likewise  delayed,  the  corpuscles  of  the  blood  have  time 
to  settle  and  form  two  distinct  layers — the  red  at  the  bottom  and  the 
white  on  top,  where  they  a})pear  as  a  grayish-white  zone,  and  con- 
stitute the  so-called  crusta  phloglstica  or  hijiammatoria.  Above  this 
we  then  see  the  plasma,  which  has  undergone  coagulation,  and  on 
top  of  it  the  serum  that  has  been  squeezed  out  from  the  clot.  The 
same  phenomenon  may  be  observed  in  human  blood  when  cooled  to 
about  0°  C,  and  from  the  extent  of  the  crusta  phlogistica  in  such 
blood  the  older  physicians  were  wont  to  draw  prognostic  conclusions 
as  to  the  course  of  the  disease. 

The  rapidity  of  coagulation  can  be  artificially  increased  and 
diminished.  By  beating  the  blood,  by  increasing  its  temperature 
a  little  beyond  that  of  the  body,  and  by  diluting  with  water  it  is 
increased ;  while  exposure  to  a  low  temperature,  the  presence  of 
much  carbon  dioxide  in  the  blood,  the  careful  lubrication  of  the 
vessel  with  vaselin  or  similar  unguents,  cause  a  retardation  of  coagu- 
lation. Its  prevention  finally  may  be  brought  about  through  influ- 
ences already  mentioned,  viz.,  by  salting  with  the  neutral  salts, 
following  the  previous  injection  into  the  body  of  albumoses  or  of 
histon,  of  diastatic  ferments,  of  extracts  of  the  mouth-parts  of  the 
leech,  after  elimination  of  the  intestinal  bloodvessels  by  ligation,  etc. 

In  addition  to  the  all)uuiins  already  considered,  viz.,  fibrinogen. 


348  THE  BLOOD. 

the  scrum -albumin,  and  serum-g'lobulin  fraction,  and  Pekelharing's 
nuoleo])rotei(l  (fibrin-ferment),  the  blood  also  contains  a  peculiar 
albuminous  substance  which  is  termed  f/hdoVm.  It  was  discovered 
by  E.  Faust  in  the  globulin  fraction  obtained  on  half-saturation  of 
the  blood-serum  with  ammonium  sulphate,  and  supposedly  occupies 
a  position  intermediate  between  the  albumins  and  collagen.  Its 
significance  is  not  known,  and  it  is  possible  that  the  substance  repre- 
sents no  ])reformed  body,  but  is  a  denaturized  globulin. 

Gliissner  and  Langstein  believe  to  have  shown  that  (ilbumoses  also 
can  occur  in  the  blood  under  normal  conditions;  that  this  is  ])ossible 
in  disease  has  long  been  known.  The  occurrence  of  still  other  al- 
buminous substances  has  from  time  to  time  been  announced,  but  has 
not  been  satisfactorily  established  (Eichholz's  mucoid,  Zanetti's 
ovomucoid-like  body,  etc.). 

Ferments. — In  addition  to  the  fibrin-ferment  the  blood-plasma 
apparently  contains  a  number  of  other  ferments.  N.  Sieber  could 
thus  demonstrate  three  different  oxidizing  ferments,  which  are  cap- 
able not  only  of  decomposing  glucose  (Lepine's  glucolytic  ferment), 
but  also  disaccharides  and  polysaccharides.  Schumm  has  demon- 
strated a  proteolytic  ferment,  others  a  lipase,  etc. 

The  remaining  solids  which  are  found  in  both  plasma  and  serum 
are,  as  has  been  pointed  out,  present  in  only  very  small  amounts. 
The  most  important  of  these  is  glucose.  Its  amount  varies  between 
1  and  1.5  pro  mille,  and  is  but  little  influenced  by  the  character  of 
the  food  unless  a  large  excess  of  carbohydrates  has  been  ingested. 
In  such  an  event  the  amount  may  increase  to  3  pro  mille,  or  even 
higher,  but  it  then  appears  also  in  the  urine,  whereby  a  further 
increase  is  prevented.  I^arger  amounts,  such  as  9  pro  mille,  are 
found  only  under  pathological  conditions. 

In  addition  to  glucose,  another  reducing  substance  is  found  in  the 
blood,  which  to  a  certain  degree  is  fermentable  and  is  soluble  in 
ether.  From  tiie  researches  of  P.  Mayer,  it  appears  that  this  sub- 
stance is  a  conjugate  glucuronate. 

Neuberg  and  Strauss  have  recently  shown  that  at  times  traces  of 
Isevulose  can  also  be  demonstrated  in  the  blood-serum,  and  that 
artificially  a  Isevulossemia  can  be  produced  in  certain  individuals 
following  the  ingestion  of  100  grammes.  The  presence  of  jecorin 
which  has  repeatedly  been  reported,  is  doubtful. 

Glycogen  is  constantly  present  in  normal  blood.  Its  amount, 
however,  is  subject  to  great  variations.  As  a  rule,  traces  only  are 
found,  but  it  may  increase  at  times  to  1.56  per  cent.,  as  calculated 
for  the  blood  as  a  whole.  Larger  amounts  are  seen  under  patho- 
logical conditions. 

Fat  is  normally  found  to  the  extent  of  from  0.2  to  0.3  ])er  cent., 
but  may  be  greatly  increased  by  the  ingestion  of  much  fatty  food, 
as  also  in  various  pathological  conditions. 

Urea  is  likewise  found  in  only  very  small  amounts  under  normal 
conditions  (0.016  to  0.020  per  cent.),  while  in  disease  much  greater 


CHEMICAL  EXAMINATION  OF  THE  BLOOD.  349 

quantities  may  be  encountered.  Ammonia  is  said  to  be  present  in 
normal  l)lood  to  the  amount  of  0.001  per  cent. 

The  further  occurrence  in  the  blood  of  soaps,  cholesterin,  and 
lecithins,  as  also  of  uric  acid,  kreatin,  carbaminic  acid,  paralactic 
acid,  hippuric  acid,  etc.,  has  been  mentioned.  In  addition  Tanella 
claims  to  have  found  small  amounts  of  phosphocarnic  acid  in  the 
blood.  All  these  bodies  are  found  in  only  extremely  small  amounts, 
and  need  not  be  considered  at  this  ]>lace.  The  pathological  constit- 
uents of  blood,  such  as  leucin,  tyrosin,  acetone,  bilirubin,  etc.,  have 
been  considered  in  preceding  chapters. 

Of  gases,  finally,  we  find  in  the  plasma  and  serum  small  amounts 
of  nitrogen  and  oxygen,  which  are  present  simply  in  solution,  and 
somewhat  larger  amounts  of  carbon  dioxide,  which  in  part  at  least 
is  more  or  less  firmly  combined  with  albumins. 

The  Leucocytes. 

The  morphological  characteristics  and  general  chemical  composi- 
tion of  the  leucocytes  have  already  been  considered  (see  pages  326 
and  329).  At  this  place  I  wish  merely  to  draw  attention  to  one 
substance  which,  according  to  Lilienfeld,  is  found  in  special 
abundance  in  the  nuclei  of  these  bodies,  and  which  has  been  termed 
nudeohiston, 

Nucleohiston. — This  substance  was  first  isolated  by  Kossel  and 
Lilienfeld  from  the  thymus  gland  of  the  calf,  but  has  since  been 
obtained  from  the  leucocytes  of  the  lymph-glands,  as  also  from  the 
splenic  cells,  the  testicular  cells,  the  spermatozoa,  and  from  the 
epithelial  lining  of  the  small  intestine.  In  all  probability  it  rej^re- 
sents  an  important  constituent  of  all  cellular  nuclei. 

Isolation. — Nucleohiston  is  most  conveniently  obtained  from  the 
leucocytes  of  the  thymus  gland.  To  this  end,  the  gland  is  carefully 
dissected  free  from  fat  and  all  larger  bloodvessels,  and  finely  hashed. 
This  mass  is  extracted  with  cold  water,  passed  through  muslin,  and 
centrifugalized.  The  aqueous  extract  is  further  filtered,  and  the 
nucleohiston  precipitated  by  the  careful  addition  of  dilute  acetic 
acid.  It  is  filtered  oif,  dissolved  in  water  with  the  aid  of  a  small 
amount  of  a  dilute  solution  of  sodium  carbonate,  reprecipitated 
with  acetic  acid,  and  purified  by  a  repetition  of  this  process.  It  is 
then  washed  with  acetic  water,  extracted  with  alcohol  and  ether, 
and  finally  dried  at  a  temperature  of  from  110°  to  115°  C.  Gam- 
gee  and  Jones  have  pointed  out  that  solutions  of  Lilienfeld's  nucleo- 
histon prepared  in  this  manner  are  very  opalescent,  but  that  this 
objection  to  the  polarimetric  examination  can  be  removed  by  ex- 
tracting the  nucleohiston  with  a  5  per  cent,  solution  of  ammonium 
acetate  and  filtering.  The  liquid  filters  very  slowly,  but  steadily. 
The  proteid  is  then  precipitated  by  pouring  tlie  solution  into  95  per 
cent,  alcohol,  after  which  it  is  washed  and  dried  with  alcohol  and 
ether. 


350  THE  BLOOD. 

Properties. — Thus  obtained,  iiucloohi.ston  represents  a  snowy- 
^vllite  fine  powder,  which  is  insoluble  in  benzol,  alcohol,  chloro- 
form, metliyl  alcohol,  ether,  and  acetic  acid,  but  is  soluble  in  water, 
glacial  acetic  acid,  concentrated  nitric  acid,  and  hydrochloric  acid, 
in  solutions  of  sodium  carbonate,  sodium  hydrate,  ammonia,  and, 
when  freshly  precipitated,  also  in  solutions  of  sodium  chloride  and 
magnesium  sulphate,  especially  in  the  presence  of  a  little  acetic  acid. 
It  has  the  properties  of  an  acid  salt,  and  on  boiling  with  water  or  on 
treating  with  baryta  water  or  very  dilute  hydrochloric  acid,  it  is 
decomposed  into  a  nuclein,  the  so-called  (cuconuclein,  and  Jdston.  It 
may  therefore  be  regarded  as  a  nucleoproteid,  but  diHers  from  most 
of  the  other  representatives  of  this  group  in  the  large  amount  of 
phosphorus — 3.025  per  cent. — which  it  vtontains.  The  histon  radicle 
possesses  marked  basic  properties,  and  readily  combines  with  acids. 
From  its  acid  solutions  it  is  precipitated  by  amnn  nia,  and  it  is  in- 
soluble in  an  excess  of  the  reagent.  The  leuconuclein,  on  the  other 
hand,  has  markedly  acid  properties.  On  treating  with  an  alcoholic 
alkali  solution  it  is  decomposed  into  an  albuminous  substance  and 
thymonucleinic  acid.  The  nucleohiston,  according  to  Garagee  and 
Jones,  is  dextrorotatory  («)D  =  -\-  37.5°. 

Elementary  analysis  of  nucleohiston  has  given  the  following 
results:  carbon,  48.46  ;  hydrogen,  7  ;  nitrogen,  16.86;  j)hosphorus, 
3.025 ;  sulphur,  0.701  ;  and  oxygen,  23.95  per  cent. 

Bang  has  lately  denied  the  existence  of  a  leuconuclein  in  the  sense 
of  Kossel.  According  to  his  researches,  Lilienfeld's  ])reparation 
represents  a  combination  of  histon  nucleinate  with  parahiston  nnclei- 
nate,  which  is  analogous  to  a  double  salt  (54  per  cent,  of  nucleinic 
acid,  30.7  per  cent,  of  histon,  and  15.3  per  cent,  of  parahiston).  It 
has  a  constant  com|5osition,  viz.,  it  is  a  hexanor7naI  acid  (adenin- 
guaninic  acid)  =  histon  -^  triadenylic  acid  =  parahiston. 

Bano^  has  pointed  out,  moreover,  that  the  results  obtained  in  the 
case  of  the  thymus  cells  are  not  directly  applicable  to  the  leucocytes 
in  general.  While  the  thymus  gland  and  lymph-glands  contain 
about  20  per  cent,  of  hi~ton  nucleinate,  no  substance  of  this  char- 
acter occurs  in  the  bone-marrow.  The  assumption  is  that  a  different 
nucleoproteid  is  here  represented. 

The  chemical  analyses  which  Lilienfeld  made  of  the  leucocytes  of 
the  thymus  gland  gave  the  following  results  : 

Water 88.51   per  cent. 

Solids    . 11.49     -     - 

Albumins ■      l-"6 

Leuconnclein       68.78 

Histon 8.67     II     II 

Lecithin       7.51 

Fats 4.02     "     " 

Cholesterin      4.40     ||     || 

Glycogen      0-80 

Nuclein -bases  as  silver  salts 15.17   -" 

Total  phcsphorus 3.01 

Total  nitrogen 15.03     "      " 


CHEMICAL   EXAMINATION  OF  THE  BLOOD.  351 

Of  albumins  proper,  there  are  said  to  be  present  in  the  leucocytes 
the  so-called  cell-globulins,  of  which  Halliburton  recognizes  two — 
one  coagulating  at  50°  C,  the  other  at  73°  C. — and  one  of  which 
is  by  some  thought  to  be  identical  with  the  fibrin  ferment ;  then 
serum-albumin,  and  a  mucinous  body,  the  so-called  hyalin  sub- 
stance of  Rovida.  These  bodies,  however,  represent  only  a  very 
small  portion  of  the  solids  of  the  leucocytes,  as  is  seen  from  Lilien- 
feld's  table,  and  it  is  doubtful  indeed  whether  their  true  chemical 
nature  has  been  sufiiciently  established. 

The  Plaques. 

Of  the  chemical  composition  of  the  plaques  little  is  known. 
According  to  Lilienfeld,  they  contain  an  albuminous  substance  and 
a  nuclein  ;  for  on  treatment  with  artificial  gastric  juice  they  can  be 
differentiated  into  a  homogeneous  portion,  which  is  subsequently 
dissolved,  and  an  insoluble  granular  portion,  which  gives  the  vari- 
ous reactions  of  the  nucleins,  and  may  be  shown  to  contain  phos- 
phorus. In  the  plaques  the  albumin  is  probably  combined  with  the 
nuclein  to  form  a  nucleoproteid,  which  may  be  identical  with  the 
nucleohiston  just  described.  According  to  Lilienfeld,  indeed  the 
plaques  must  be  regarded  as  nuclear  derivatives,  and  he  has  accord- 
ingly termed  them  the  nuclein  platelets  of  the  blood. 

The  Red  Corpuscles. 

The  red  corpuscles  of  the  blood,  as  has  been  mentioned,  owe  their 
color  to  the  presence  of  hsemoglobin  or  its  oxygen  compound,  oxy- 
hsemoglobin.  This  may  be  extracted  by  diluting  with  water,  by 
alternate  freezing  and  thawing,  by  shaking  with  ether,  chloroform, 
etc.  The  blood  is  thereby  rendered  lake-colored,  and  on  microscopi- 
cal examination  it  will  be  observed  that  instead  of  the  original  cor- 
puscles, so-called  blood-shadows  are  now  found.  These  are  colorless 
ring-like  bodies,  and  constitute  the  stroma  of  the  red  corpuscles. 
In  the  circulating  blood  the  dissolution  of  the  haemoglobin  is  pre- 
vented by  the  presence  of  large  amounts  of  sodium  chloride.  Such 
blood  is  said  to  be  hi/perisotonic — /.  e.,  it  contains  more  sodium 
chloride  than  is  necessary  to  prevent  the  dissolution  of  the  coloring- 
matter  from  its  corpuscles.  Within  the  corpuscles  the  hemoglobin 
is,  however,  supposedly  not  present  in  the  free  state,  but  in  com- 
bination with  some  other  substance,  such  as  lecithin  ;  and  Hoppe- 
Seyler  accordingly  distinguishes  between  the  so-called  arterin  and 
phlehin,  which  represents  the  lecithin  compound  of  oxy haemoglobin 
and  haemoglobin  respectively. 

As  has  been  mentioned,  the  red  corpuscles  represent  nearly  one- 
half  of  the  liquid  blood.  They  contain  about  57,7  per  cent,  of 
water  and  40,5  ])er  cent,  of  oxy haemoglobin,  while  the  constituents 
of  the  stroma  inclusive  of  mineral  salts  amount  only  to  about  1,9  per 
cent,     AmoncT  these  constituents  Halliburton's  cell-globulin  is  said 


352  THE  BLOOD. 

to  be  most  abundant;  in  addition  we  find  traces  of  lecithin,  choles- 
terin,  and  nucleo-albnmin,  while  serum-albumin  and  albumoses  are 
apparently  absent.  In  the  nucleated  red  corpuscles  of  birds  we 
further  meet  w^ith  the  integral  constituents  of  the  nuclei,  among 
which  Lilienfeld's  nucleohiston  probably  always  prevails.  Its  basic 
constituent,  histon,  Mas  first  discovered  by  Kossel  in  the  red  cor- 
puscles of  the  goose. 

An  idea  of  the  mineral  constituents  of  the  red  corpuscles,  viz., 
their  stroma,  may  be  had  from  the  following  table,  which  is  taken 
from  C.  Schmidt,  and  calculated  for  100  parts  of  the  moist  cor- 
puscles. 

Potassium  chloride      3.68 

Sodium  cliloride traces. 

Potassium  sulpliate      0.13 

Potassium  i)hosi)hate 2.34 

Sodium  phosphate 0.63 

Calcium  phosphate       0.09 

Magnesium  phospliate 0.06 

The  iron  of  the  haemoglobin  is  not  included  in  this  table ;  in  man 
it  varies  between  0.506  and  0.537  pro  mille.  In  addition  traces  of 
copper  are  not  infrequently  met  w4th,  and,  as  will  be  seen  later,  this 
element  in  some  of  the  invertebrate  animals  apparently  takes  the 
place  of  iron  in  the  coloring-matter  of  the  blood. 

Isolation  of  the  Red  Corpuscles  of  the  Blood. — To  isolate  the 
red  corpuscles,  the  blood  is  defibrinated  by  beating,  diluted  with  ten 
times  its  volume  of  a  1  per  cent,  solution  of  sodium  chloride,  and 
passed  through  a  muslin  filter.  On  subsequent  centrifugation  and 
repeated  washing  with  the  salt  solution,  they  are  then  freed  from  the 
serum,  and  may  now  be  collected  on  a  paper  filter,  after  the  previous 
addition  of  a  large  amount  of  alcohol.  Any  fats,  lecithins,  or 
cholesterins  that  may  be  present  are  extracted  with  warm  alcohol 
and  ether,  when  the  corpuscles  can  be  dried  and  weighed.  To 
isolate  the  stromata,  the  mass  of  corpuscles,  when  freed  from  serum, 
is  shaken  with  five  or  six  times  its  volume  of  water  and  a  small 
amount  of  ether.  The  mixture  is  then  centrifugalized,  and  is  thus 
separated  from  the  leucocytes.  The  stromata  are  now  precipitated 
by  adding  a  few  drops  of  a  1  per  cent,  solution  of  acid  sodium 
phosphate,  until  the  liquid  has  almost  assumed  the  consistence  of 
the  original  blood.  They  are  then  collected  on  a  filter,  quickly 
washed  with  water,  and  may  now  be  dissolved  in  a  5  per  cent,  solu- 
tion of  magnesium  sulphate. 

Hsemoglobin  and  Its  Derivatives. 

Haemoglobin. — The  haemoglobin  is  the  coloring-matter  of  the 
red  corpuscles,  and  is  present  in  these  in  combination  with  an- 
other body,  which  may  be  a  lecithin,  as  the  phlebin  of  Hoppe- 
Seyler,  while  the  corresponding  compoimd  of  oxyhasraoglobin  is 
termed    arterin.     Of    the    nature    of    these    compounds,    however, 


CHEMICAL  EXAMINATION  OF  THE  BLOOD.  353 

but  little  is  known,  and  it  has  not  even  been  definitely  ascertained 
that  the  pairling  of  the  coloring-matter  is  really  a  lecithin. 

Hsenioglobin  or  its  oxy-compound  occurs  widely  distributed  in 
the  animal  world,  and  is  found  not  only  in  the  vertebrates,  but  also 
in  many  of  the  invertebrates.  But  while  in  the  former  it  is  con- 
tained in  definite  cellular  elements  of  the  blood,  it  may  also  occur  as 
such  among  certain  invertebrate  animals.  Closely  related  to  it  is 
the  so-called  oxyhcemocyanin,  which  is  found  in  certain  arthropods 
and  molluscs,  and  in  which  the  iron  is  apparently  replaced  by 
copper.  Then  again  we  find  among  invertebrate  animals  various 
violet  and  purplish-red  pigments,  the  so-called  jioridins,  which  are 
likewise  to  be  classed  with  haemoglobin,  and  as  we  have  previously 
seen,  a  genetic  relationship  apparently  also  exists  between  haemo- 
globin and  the  chlorophyl  of  plants.  These  various  pigments  are 
collectively  spoken  of  as  respiratory  pigments,  as  they  are  intimately 
concerned  in  tlie  transportation  of  the  oxygen  of  the  air  to  the 
various  tissues  of  the  body,  and  in  the  removal  of  the  carbon 
dioxide  which  results  as  a  product  of  cellular  metabolism. 

Outside  of  the  blood  haemoglobin  is  found  also  in  striped  and  un- 
striped  muscle-tissues,  and  under  pathological  conditions  it  may 
appear  in  the  urine  as  such.  Diiferent  haemoglobins  apparently 
exist.  It  is  hence  impossible  to  give  a  definite  formula  which  ex- 
presses the  constitution  of  all.  An  idea  of  their  quantitative  ele- 
mentary composition  may  be  had  from  the  accompanying  table  : 

Carbon.  Hydrogen.  Nitrogen.  Oxygen.  Sulphur.  Phosphorus.  Iron, 

Horse 54.87  6.97  17.31  19.730  0.650           .    .  0.470 

Dog 54.57  7.22  16.38  20.930  0.568           .    .  0.336 

Pig      54.71  7.38  17.43  19.602  0.479           .    .  0.399 

Guinea-pig     ....  54.12  7.36  16.78  20.680  0.580           .    .  0.480 

Squirrel 54.09  7.39  16.09  21.440  0.400           .    .  0.590 

Goose      54.26  7.10  16.21  20.690  0.540  0.770  0.430 

Chicken      52.47  7.19  16.45  22.500  0.857  0.197  0.335 

The  size  of  the  haemoglobin  molecule  is,  like  that  of  all  albu- 
minous substances,  very  large.  For  that  of  dog's  blood  Hiifner 
obtained  the  figure  14,129,  which  would  correspond  to  the  formula 
Q36Hio25Nig4FeS30igi.  It  thus  contains  three  atoms  of  sulphur  for 
one  atom  of  iron,  while  the  haemoglobin  of  the  horse  and  pig  has 
only  two  atoms  of  sulphur  for  one  atom  of  iron.  Of  interest 
further  is  the  presence  of  phosphorus  in  the  haemoglobin  of  the 
goose  and  chicken.  Whether  this  forms  an  integral  component  of 
the  haemoglobin  molecule,  however,  is  questionable,  and  it  is  quite 
possible  that  its  presence  is  owing  to  a  contamination  of  the  coloring- 
matter  with  nucleinic  acid  derived  from  the  nuclei  of  the  red 
corpuscles. 

Structurally,  haemoglobin  must  be  regarded  as  a  proteid,  viz.,  as  a 
compound  of  an  albuminous  radicle  with  another  complex  organic 
radicle.  This  other  radicle  is  here  an  iron-containing  pigment, 
which  may  be  separated  from  its  albuminous  pairling,  and  is  termed 

23 


354  THE  BLOOD. 

hoemochromogen  (see  below),  while  the  albuminous  substance  is 
known  as  glohin.  These  two  substances  are  apparently  united  in 
the  haemoglobin  molecule,  through  an  additional  radicle,  which  is  as 
yet  unknown.  Haemoglobin,  like  the  nucleoproteids,  is  dextrorota- 
tory. 

Globin  is  a  histon,  and  accordingly  presents  the  following  charac- 
teristic reactions  :  it  is  precipitated  by  ammonia  from  its  solutions  in 
dilute  hydrochloric  acid,  and  is  insoluble  in  an  excess  of  the  reagent. 
With  concentrated  nitric  acid  it  is  thrown  down  in  the  cold,  but  not 
from  its  heated  solutions.  Under  certain  conditions  it  can  be  coagu- 
lated on  boiling,  but,  unlike  the  other  coagulable  albumins,  its  coagu- 
late is  readily  soluble  in  acids.  It  contains  5-4.97  per  cent,  of  carbon, 
7.2  per  cent,  of  hydrogen,  16.89  per  cent,  of  nitrogen,  and  0.42  per 
cent,  of  sulphur.  In  its  behavior  toward  polarized  light  globin 
behaves  like  a  true  albumin — i.  e.,  it  is  kevorotatory.  The  amount 
of  globin  which  can  be  obtained  from  the  haemoglobin  molecule  is 
quite  large,  and  according  to  Schultz  amounts  to  86.5  per  cent., 
while  4.2  per  cent,  only  is  represented  by  the  pigment  itself. 

Isolation. — A  solution  of  oxyhaemoglobin  in  water,  prepared  at 
a  temperature  of  40°  C,  is  treated  with  dilute  hydrochloric  acid 
until  the  red  color  changes  to  brown.  This  mixture  is  extracted 
with  80  per  cent,  alcohol  (one-fifth  volume)  and  ether  (one-half 
volume)  until  the  ether  takes  up  no  more  coloring-matter.  The 
resulting  aqueous-alcoholic  solution  is  precipitated  with  ammonia, 
filtered,  and  the  precipitate  dissolved  in  \ery  dilute  acetic  acid.  On 
filtering,  the  globin  is  again  precipitated  with  ammonia  and  col- 
lected on  a  silk  filter.  After  washing  with  absolute  alcohol,  then 
with  water,  again  with  alcohol,  and  finally  with  ether,  the  substance 
is  dried  first  in  the  air  and  then  at  a  temperature  of  100°  C.  The 
resulting  material  constitutes  pure  globin,  as  a  yellowish  loose 
powder,  which  is  not  especially  hygroscopic. 

Hsemochromogen. — The  isolation  of  ha?mochromogen  is  rather 
difficult,  owing  to  the  avidity  with  which  it  combines  with  oxygen  to 
form  hcematin  in  alkaline  solution.  Hoppe-Seyler,  however,  suc- 
ceeded in  obtaining  the  substance  in  crystalline  form,  by  heating 
haemoglobin  with  sodium  hydrate  solution  in  an  atmosphere  of  hy- 
drogen. In  acid  solutions  haemochromogen  gradually  loses  its  iron 
and  is  converted  into  hfematoporphyrin.  In  alkaline  solution  it 
presents  a  Ijeautiful  cherry-red  color,  and  on  spectroscopic  examina- 
tion gives  two  bands  of  absorption.  One  of  these  is  very  intense, 
and  located  between  D  and  E,  nearer  D,  while  the  other  is  not  so 
dark,  but  wider,  and  found  about  E  and  extending  beyond  b.  To 
demonstrate  the  spectrum  of  hfemochromogen,  bloody  fluid  is  mixed 
■with  a  solution  of  sodium  hydrate,  when  the  resulting  hsematin  is 
reduced  Avith  ammonium  sulphide,  or  Stokes  reagent,  viz.,  an  am- 
moniacal  solution  of  ferrous  tartrate,  or  stannous  chloride. 

The  haemochromogen  radicle,  as  has  been  stated,  represents  the 
pigmented  group  of  the  haemoglobin  molecule,  and  to  its  presence 


CHEMICAL  EXAMINATION  OF  THE  BLOOD.  355 

the  power  of  lifemofjlobin  to  combine  with  oxygen,  carbon  dioxide, 
and  other  gases,  is  unquestionably  due. 

Hcemoglohin  itself  can  be  obtained  in  crystalline  form,  and  is 
characterized  by  the  great  resistance  which  it  offers  to  putrefaction 
and  tryptic  decomposition.  It  is  stated  that  even  after  the  lapse  of 
years  decomposed  blood  contains  its  haemoglobin  as  such,  and  that 
on  shaking  with  air  it  may  again  be  transformed  into  pure  oxy- 
hsenioglobin.  Its  solutions  present  a  beautiful  purplish-red  color, 
and  on  spectroscopic  examination  gives  rise  to  a  single  band  of 
absorption,  w^hich  lies  between  D  and  E  and  extends  slightly  to  the 
left  beyond  D.  This  is  most  conveniently  shown  by  taking  a  solu- 
tion of  oxyhtemoglobin,  and  reducing  this  with  ammonium  sulphide 
or  Stokes'  fluid,  as  directed  above. 

Most  important  is  the  avidity  with  which  haemoglobin  combines 
with  various  gases,  and  upon  this  characteristic  indeed  its  chief 
function,  as  a  respiratory  pigment,  is  based.  This  property,  as  has 
been  stated,  is  referable  to  the  chromogcn  radicle,  and  more  partic- 
ularly to  the  iron,  which  it  contains.  Every  atom  of  this  is  capable 
of  combining  with  one  molecule  of  oxygen  or  of  carbon  dioxide, 
and  in  this  form  largely  the  oxygen  of  the  air  is  carried  to  the  vari- 
ous tissues  of  the  body,  and  the  carbon  dioxide  removed.  In  the 
circulating  blood  we  accordingly  find  only  relatively  small  amounts 
of  free  haemoglobin,  and  in  arterial  blood  its  oxy-compound  is 
almost  exclusivelv  encountered. 

The  amount  of  haemoglobin  which  is  contained  in  human  blood, 
either  as  such  or  in  combination  with  oxygen  or  carbon  dioxide,  is 
about  14  per  cent.,  but  subject  to  certain  variations,  even  in  health, 
while  in  disease  still  greater  deviations  from  the  average  normal 
amount  are  observed.  A  great  diminution  may  here  occur,  and  is 
most  marked  in  chlorosis  and  pernicious  anaemia,  in  which  the  per- 
centage may  fall  as  low  as  2.35. 

As  the  isolation  of  the  haemoglobin  from  the  blood  resolves 
itself  into  the  isolation  of  its  oxy-compound,  this  will  be  considered 
together  with  its  quantitative  estimation  under  that  heading. 

Oxyhaemoglobin. — Oxyhaemoglobin  is  the  oxy-compound  of  haemo- 
globin, and  differs  from  its  mother-substance  in  containing  two 
atoms  more  of  oxygen,  which  are  bound  to  the  one  atom  of  iron, 
than  are  present  in  the  haemochromogen  radicle.  In  this  manner, 
however,  the  haemochromogen  is  converted  into  haematin,  and  we 
may  therefore  say  that  oxyhaemoglobin,  in  contradistinction  to 
haemoglobin,  consists  of  the  globin  radicle  united  by  some  unknown 
group  to  a  haematin  radicle  (see  also  page  358). 

Haematin. — In  accordance  with  the  above  considerations,  we 
find  that  on  decomposition  of  oxyhaemoglobin  haematin  is  obtained 
instead  of  haemochromogen.  This  latter,  indeed,  is  at  once  trans- 
formed into  haematin  on  exposure  to  oxygen,  and,  as  we  have  seen, 
the  haematin  is  correspondingly  reconverted  into  haemochromogen  by 
treating  with   reducing  agents.     The  decomposition   of  oxyhsemo- 


356  THE  BLOOD. 

globin  with  the  formation  of  hoematin  can  be  readily  effected  by 
heating  its  solutions  to  a  temperature  of  80°  C,  bv  treating  with 
dilute  mineral  acids,  or  with  stronger  solutions  of  the  alkaline 
hydrates,  as  also  by  peptic  or  tryptic  digestion.  To  obtain  the  sub- 
stance in  a  pure  state,  however,  it  is  best  to  start  with  its  hydro- 
chlorate  anhydride,  hcemin,  w^hich  can  be  readily  obtained  in  crystal- 
line form.  To  this  end,  oxyhaemogloliin  is  treated  with  a  trace  of 
sodium  chloride  and  dissolved  in  glacial  acetic  acid.  On  heating, 
the  hsemin  is  precipitated  in  the  form  of  very  characteristic,  drawn- 
out,  rhombic  platelets,  which  are  insoluble  in  water,  alcohol,  and 
ether,  with  difficulty  so  in  glacial  acetic  acid  and  in  dilute  mineral 
acids,  but  are  readily  soluble  in  dilute  alkaline  solutions  and  acid 
alcohol.  The  crystals  are  collected  on  a  filter,  washed  with  alcohol 
and  ether,  and  are  thus  obtained  in  pure  form.  To  prepare  the 
haematin,  the  haemin  is  dissolved  in  a  dilute  solution  of  sodium 
hydrate  and  supersaturated  with  dilute  hydrochloric  acid.  The  sub- 
stance is  thus  precipitated  in  the  form  of  brownish  flakes,  which  are 
w^ashed  free  from  chlorides  and  dried  at  120°  C.  Haematin,  in  con- 
tradistinction to  oxyhsemoglobin  and  hfemin,  is  non-crystallizable. 
Its  solubility  is  essentially  the  same  as  that  of  hsemin.  In  acid  solu- 
tion both  hsematin  and  hsemin  show  a  well-defined  spectral  band 
between  C  and  D.  Between  D  and  F  a  second  band  is  seen,  which 
is  much  wider  but  less  sharply  defined  than  the  first.  By  diluting 
the  solution  this  band  may  be  resolved  into  three  bands,  of  which 
one  is  located  between  b  and  F,  near  F ;  another,  between  D  and  E, 
nearer  E ;  and  a  third  faint  band  between  D  and  E,  nearer  D.  As 
a  rule,  however,  only  two  bands  are  seen.  The  alkaline  solutions  of 
hsematin  are  distinctly  dichrotic  and  give  only  one  band  of  absorp- 
tion, the  greater  portion  of  which  lies  between  C  and  D,  and  extends 
slightly  beyond  T). 

The  basis  of  the  hsematin  molecule  is  supposedly  a  methyl-propyl- 

pyrrol : 

CHo— C C-C3H7 

II  II  (C-sHigN) 

HC\     /CH 
N 
H 

This  is  probably  the  mother-substance  of  the  hsematinic  acids 
which  Kiister  obtained  from  hsematin  on  careful  oxidation,  viz.,  the 
dibasic  acid  C^HgNO^,  and  the  tribasic  acid  CgHgO^  which  results 
from  the  former.  The  structural  formuhe  of  these  acids,  according 
to  Kiister,  are  the  following  : 

CH3— C==C— CHj.  CH2— COOH 

I     I  (CfiH^NH,) 

0C\   /CO 

H 

CH3— C=^C-CH2— CH2— COOH 

■   I      I  (CaHPs) 

0C\  /CO 


CHEMICAL  EXAMINATION  OF  THE  BLOOD.  357 

On  treating  with  concentrated  sulphnric  acid,  with  hydrochloric 
acid,  or  with  glacial  acetic  and  hydrobromic  acids,  hsematin  loses  its 
iron,  and  on  the  subsequent  addition  of  water  is  transformed  into 
hfematoporphyrin,  whicli  is  isomeric  with  bilirubin.  This  change  is 
expressed  in  the  equation  : 

C32H3,N,0,Fe    +    2H,0    -    Fe    =    Cg.HgeNA 
Hsematin  Bilirubin 

(hsematoporphyrin). 

Bilirubin,  according  to  Kiister,  yields  the  same  acids  on  oxida- 
tion as  ha^matin  or  at  least  isomeric  bodies.  The  intimate  connec- 
tion existing  between  the  blood-pigment  and  bile-pigment,  and 
through  this  with  the  urinary  pigment,  is  thus  satisfactorily  estab- 
lished. 

On  reduction  with  tin  and  hydrochloric  acid  in  alcoholic  solution 
and  the  subsequent  addition  of  caustic  alkali  in  excess  hsematin 
gives  rise  to  skatol.  On  intense  oxidation  with  ammonium  persul- 
phate cyanic  acid  and  succinic  acid  result. 

As  hrematin  is  a  decomposition-product  of  oxyhsemoglobin  it 
is  not  found  as  such  in  the  circulating  blood.  It  is  said  to  occur  in 
the  urine,  however,  in  cases  of  poisoning  with  arsenious  hydride. 
In  the  stools  it  is  found  after  hemorrhages  into  the  stomach  or  the 
upper  portion  of  the  small  intestine,  and  also  after  the  ingestion  of 
large  amounts  of  red  meats.  In  such  cases,  of  course,  its  origin  is 
referable  to  the  decomposition  of  hsemoglobin  through  the  agency  of 
the  gastric  and  pancreatic  juices. 

Haemin. — AVhile  formerly  hsemin  was  regarded  as  a  salt  of  hsema- 
tin,  it  is  now  undoubted  that  its  chlorine  is  present  in  combination 
with  carbon  or  iron.  By  long-continued  washing  with  hot  water 
the  chlorine  can  be  almost  entirely  removed,  viz.,  replaced,  by 
hydroxyl.  It  is  also  generally  accepted  that  the  iron  can  be  in  part 
split  off  on  heating  with  alkalies.  Very  curiously  elementary  analy- 
sis of  the  substance  has  given  the  same  results  to  the  same  observer, 
but  different  results  to  different  observers.  Nencki  has  shown  that 
this  is  owing  to  the  fact  that  hsemin  contains  two  hydroxyl  groups, 
and  that  the  substance  not  only  forms  ethers  with  acid  and  alkyl 
radicles  with  great  readiness,  but  also  addition-products  with  indif- 
ferent compounds.  The  most  probable  formula  of  the  hsemin  which 
can  be  obtained  from  hremoglobin  is  €3211320^^401  Fe  (Nencki).  Its 
preparation  has  been  described  above. 

Isolation  of  Oxyhaemoglobin. — Oxyhfemoglobin  in  crystalline  form 
is  best  obtained  from  the  red  corpuscles  of  the  horse  or  dog, 
according  to  the  following  method  :  the  blood  is  first  rendered 
uncoagulable  by  treating  with  ammonium  oxalate  (0.06-0.1  per 
cent.),  and  is  then  centrifugalized  to  effect  the  separation  of  the  cor- 
puscles. This  mass,  when  freed  from  plasma  by  siphonage,  is  treated 
with  twice  its  volume  of  water  and  placed  on  ice.  The  resulting 
fluid  is  now  mixed  with  an  equal  volume  of  a  saturated  solution  of 
ammonium  sulphate  that  has  likewise  been  cooled  to  a  low  tempera- 


358  THE  BLOOD. 

ture.  This  mixture  is  also  placed  on  ice  until  the  jirccipitate  of 
globulins,  which  is  referable  to  remaining  plasma,  has  settled.  On 
filtering  in  the  refrigerator  a  perfectly  clear  dark-red  filtrate  is 
obtained,  which  contains  the  greater  portion  of  the  coloring-matter. 
If  now  the  solution  is  brought  to  the  temperature  of  the  room, 
the  separation  of  crystalline  oxyhsemoglobin  begins,  and  may  be 
hastened,  if  necessary,  by  the  further  addition  of  a  small  amount 
of  the  ammonium  sulphate  solution.  After  a  few  days  the  process 
is  completed,  when  the  crystals  are  filtered  off  through  a  Biichner 
filter  by  the  aid  of  a  suction  pump,  and  are  partially  freed  from  the 
mother-liquor  by  pressing  between  filter-paper.  The  substance  is 
then  purified  by  recrystallization.  To  this  end,  the  crystalline 
mass  is  dissolved  in  water,  reprecipitated  by  the  addition  of  an  equal 
volume  of  the  ammonium  sulphate  solution,  and  so  on,  until  the 
required  degree  of  purity  has  been  attained.  Adhering  ammonium 
sulphate  is  finally  removed  mechanically.  To  preserve  the  sub- 
stance, it  is  dried  in  a  vacuum  or  at  a  low  temperature  over  sulphuric 
acid,  and  is  then  quite  stable. 

The  ease  with  which  oxyhsemoglobin  can  be  brought  to  crystal- 
lization diifers  with  different  animals.  In  guinea-pigs,  squirrels,  and 
rats  it  is  most  pronounced ;  and  it  is  here  only  necessary  to  mix  a 
few  drops  of  the  blood  with  an  equal  amount  of  water,  when  the 
process  may  be  directly  observed  with  the  microscope.  Human 
blood,  as  also  that  of  the  pig  and  the  ox,  is  much  more  refractory, 
and  is  not  well  adapted  for  the  preparation  of  the  pigment  in  its 
crystalline  state. 

The  form  of  the  crystals  varies  in  different  animals.  We  thus 
find  hexagonal  platelets  in  the  squirrel,  rhombic  tetrahedra  in 
the  guinea-pig  and  various  birds,  rhombic  needles  in  man,  etc. 

In  its  chemical  behavior  oxy haemoglobin  manifests  its  albuminous 
nature.  It  is  thus  coagulated  on  boiling,  and  on  treating  with 
absolute  alcohol  and  most  of  the  salts  of  the  heavy  metals.  It  is  to 
be  noted,  however,  that  the  process  of  coagulation  is  associated  with 
the  decomposition  of  the  compound  into  hoematin  and  globin,  which 
latter  is  precipitated  in  its  coagulated  state.  From  its  solutions 
the  substance  is  thrown  down  by  half-saturation  with  ammonium 
sulphate.  On  reduction  with  ammonium  sulphide,  with  Stokes' 
reagent,'  or  during  the  process  of  putrefaction,  it  is  transformed 
into  hsemoglobin.  Other  reducing  agents,  such  as  sodium  hydro- 
sulphite,  give  rise  to  the  formation  of  so-called  pseudohfemoglobin, 
which  apparently  stands  midway  between  oxyhemoglobin  and 
hsemoglobin  in  containing  less  oxygen  than  the  former,  but  more 
than  the  latter.  Its  spectrum,  however,  is  the  same  as  that  of  the 
completely  reduced  hajnioglobiu. 

In  sufficiently  dilute  solution  oxyhsemoglobin  shows  two  bands 
of  absorption  between  D  and  E.  The  one  to  the  left,  which  is  not 
so  wide  as  the  other,  but  darker  and  more  sharply  defined,  borders 
on  D,  while  the  second  lies  at  E. 


CHEMICAL   EXAMINATION  OF  THE  BLOOD.  359 

The  Quantitative  Estimation  of  Oxyhsemoglobin. — This  is  best 
accomplished  by  Hoppe-Seyler's  method,  which  is  based  upon 
the  comparison  of  a  given  amount  of  diluted  blood  with  a  stand- 
ard solution  of  crystallized  oxyhfemoglobin.  This  solution  is 
prepared  by  dissolving  2  grammes  of  the  pure  coloring-matter  in 
50  c.c.  of  distilled  water.  The  oxyhaemoglobin  is  then  transformed 
into  carbon  monoxide  haemoglobin  by  passing  a  current  of  the  gas 
through  the  solution  to  saturation.  It  is  then  stored  in  drawn- 
out  and  sealed  glass  tubes,  such  that  each  tube  contains  about 
6  c.c.  The  contents  of  each  tube,  when  diluted  M'ith  ten  times  its 
volume  of  water,  will  then  represent  a  0.2  per  cent,  solution  of  the 
oxy  haemoglobin. 

A  carefully  measured  or  weighed  amount  of  blood,  not  exceed- 
ing 0.5  c.c,  is  now  diluted  with  water  that  has  been  saturated  with 
carbon  monoxide  to  exactly  5  c.c.  A  small  drop  of  a  very  dilute 
solution  of  sodium  hydrate  is  added  if  necessary  to  remove  any 
turbidity  that  may  exist.  This  solution  is  further  saturated  with  car- 
bon monoxide,  and  freed  from  fibrin  by  filtration.  The  filtrate  should 
measure  exactly  4  c.c.  The  comparison  of  the  two  solutions,  and 
the  further  dilution  of  tlie  blood  with  carbon  monoxide  water  then 
takes  place  in  the  so-called  double  pipette  of  Hoppe-Seyler.  The 
color  of  the  two  solutions  is  here  equalized,  and  the  amount  of 
hseraoglobin  present  in  the  specimen  of  blood  calculated  from  the 
degree  of  dilution. 

Example. — Suppose  that  we  started  with  0.5  gramme  of  blood,  and 
that  the  standard  solution  contained  0.002  gramme  of  oxyhaemoglo- 
bin  in  the  cubic  centimeter.  The  4  c.c.  of  the  diluted  and  filtered  blood 
are  further  diluted  in  the  pipette  to  22  c.c,  which  corresponds  to  a 
total  solution  of  27.5  c.c.  for  the  total  5  c.c.  of  the  first  dilution.  In 
these  27.5  c.c,  which  represent  the  original  0.5  gramme  of  blood, 
there  will  consequently  be  27.5  X  0.002  =  0.0550  gramme  of 
haemoglobin.     The  percentage  will  accordingly  be  11  per  cent. 

In  the  clinical  laboratory  other  forms  of  apparatus  are  in  use, 
such  as  the  hccmometers  of  Dare  and  Fleischl,  and  the  hcemoglobin- 
ometer  of  Gowers.  In  the  first  the  color  of  the  blood  is  compared  with 
that  of  a  glass  wedge  that  has  been  stained  with  the  golden  purple 
of  Cassius.  The  same  principle  holds  good  for  the  second.  In  the 
third  a  standard  solution  of  carmin  and  picric  acid  is  employed. 

The  spectro-photometric  determination  of  the  blood  coloring- 
matter  is  not  described  at  this  place.  It  is  undoubtedly  the  most 
exact,  but  necessitates  the  use  of  costly  apparatus,  which  will  be 
found  in  only  few  laboratories. 

It  has  been  pointed  out  that  haemoglobin  is  characterized  by  the 
readiness  with  which  it  combines  with  certain  gases,  and  we  have 
just  considered  the  most  important  of  these  compounds.  Other 
compounds  of  this  character  are  carbon  dioxide  haemoglobin,  carbon 
monoxide  haemoglobin,  nitric  oxide  haemoglobin,  and  cyanhaemo- 
globin. 


360  THE  BLOOD. 

Carbon  Dioxide  Haemoglobin. — Three  different  forms  are  said  to 
exist,  which  have  been  respectively  termed  a,  /9,  and  j  carboluvmo- 
fllohin,  but  they  are  comparatively  little  known.  According  to 
Bohr,  the  carbon  dioxide  in  these  compounds  is  united  with  the 
all)uminous  radicle  of  the  hajraoglobin,  while  the  oxygen  of  oxy- 
hiemoglobin  is  combined  with  the  pigmented  group.  He  accordingly 
finds  that  when  a  solution  of  haemoglobin  is  shaken  with  a  mixture 
of  oxygen  and  carbon  dioxide  both  of  these  gases  are  taken  up  inde- 
pendently of  each  other. 

Carbon  Monoxide  Haemoglobin. — This  compound  results  from 
the  union  of  one  molecule  of  haemoglobin  with  one  molecule  of  carbon 
monoxide,  and  is  characterized  by  its  greater  stability  as  compared 
with  oxyhsemoglobin.  The  carbon  monoxide  is  in  this  case  united 
with  the  pigmented  radicle  of  the  haemoglobin,  and  may  be  split  oif 
in  this  combination  as  carbon  monoxide  hajmochromogen.  Like  the 
native  htemochromogen,  the  carbon  monoxide  compound  can  be 
obtained  in  crystalline  form,  and  on  exposure  to  the  air  is  likewise 
transformed  into  heematin.  Under  the  same  conditions  the  haemo- 
globin compound  is  gradually  reconverted  into  oxy haemoglobin. 

Blood  containing  carbon  monoxide  haemoglobin  is  characterized 
by  its  cherry-red  color,  its  resistance  to  putrefactive  changes  in  the 
absence  of  oxygen,  and  by  its  spectrum.  This  is  similar  to  that  of 
oxyhsemoglobin,  but  its  two  bands  of  absorption,  between  D  and  E, 
are  placed  rather  nearer  the  violet  end  of  the  spectrum.  Unlike  the 
spectrum  of  oxyhsemoglobin,  however,  that  of  the  carbon  monoxide 
compound  is  not  changed  to  the  haemoglobin  spectrum  on  treating 
with  reducing  agents.  Should  oxyhaemoglobin  be  simultaneously 
present,  a  mixed  spectrum  of  the  two  substances  is  obtained. 

Such  blood,  moreover,  when  treated  with  double  its  volume  of  a 
solution  of  sodium  hydrate  (sp.  gr.  1.3),  is  not  changed  to  a  dirty 
brownish  mass,  with  a  tint  of  green,  as  with  normal  blood,  but 
presents  a  beautiful  red  color,  which  changes  to  brown  only  on 
standing. 

In  its  crystalline  state  carbon  monoxide  haemoglobin  may  be 
obtained  by  saturating  a  sufficiently  concentrated  solution  with  car- 
bon monoxide  and  cooling  the  mixture  to  0°  C,  when  one-fourth  of 
its  volume  of  cooled  alcohol  is  added.  On  standing  in  the  refrigera- 
tor the  substance  separates  out  in  the  form  of  bluish-red  crystals, 
which  are  isomorphous  with  those  of  oxyhaemoglobin,  but  much 
more  stable. 

Nitric  Oxide  Haemoglobin. — This  compound  is  more  stable  even 
than  carbon  monoxide  haemoglobin,  and,  like  this,  may  be  obtained 
in  crystalline  form.  Its  spectrum  is  similar  to  that  of  carbon  mon- 
oxide haemoglobin.  The  bands,  however,  are  less  sharply  defined 
and  paler  than  those  of  that  compound,  and,  like  these,  do  not  dis- 
appear on  the  addition  of  a  reducing  agent.  The  substance  is  met 
with  in  poisoning  by  the  gas  in  question. 

Cyanhaemoglobin. — This  has  been  obtained  in  crystalline  form 


CHEMICAL  EXAMINATION  OF  THE  BLOOD.  361 

by  V.  Zeynek  ;  it  contains  one  molecule  of  hydrocyanic  acid  or  the 
univalent  cyanogen  radicle  in  firm  combination.  Its  toxicity  is 
comparatively  slight. 

Kathaemoglobin. — This  term  has  been  applied  by  Van  Klaveren 
to  a  decomposition-product  of  haemoglobin  which  results  on  bailing 
a  mixture  of  defibrinated  blood  with  an  alcoholic  solution  of  sodium 
hydrate.  Unlike  haematin,  it  is  not  a  decomposition-product  that 
is  free  from  albumin,  but  is  a  proteid  which  is  still  quite  closely 
related  to  haemoglobin.  Arnold  first  described  this  substance  as 
neutral  h;^matin.  It  contains  somewhat  less  iron  than  haemoglobin, 
a  portion  being  split  off  during  its  formation  as  a  water-soluble 
organic  compound  which  escapes  on  dialysis. 

Methsemoglobin. — Methsemoglobin  is  a  pigment  which  normally 
does  not  occur  in  the  blood,  but  is  found  after  the  ingestion  of  large 
amounts  of  potassium  chlorate,  antifebrin,  potassium  permanganate, 
turpentine,  kairin,  thallin,  following  the  inhalation  of  nitrite  of 
amyl,  ether,  etc.  It  is  encountered  also  in  hemorrhagic  transudates 
and  cystic  fluids,  and  may  occur  in  the  urine  when  methsemoglobin- 
aemia  exists. 

The  elementary  composition  of  metha?moglobin  is  the  same  as 
that  of  oxyhemoglobin,  but  its  molecular  structure  is  manifestly 
different,  as  in  a  vacuum  it  does  not  give  up  its  oxygen.  On 
treating  with  reducing  agents  or  on  exposure  to  putrefactive  organ- 
isms, in  the  absence  of  oxygen,  it  is  converted  into  haemoglobin. 
When  oxyhaemoglobin  is  decomposed  with  dilute  acids  or  alkalies, 
methaemoglobin  is  formed  at  some  stage  of  the  process,  and  precedes 
the  formation  of  hsematin.  During  the  preservation  of  oxyhaemo- 
globin in  the  dry  state,  moreover,  a  partial  transformation  into 
methfemoglobin  is  very  likely  to  occur.  The  substance  is  crystal- 
lizable,  and  may  be  obtained  in  this  form  by  treating  a  concentrated 
solution  of  oxyhaemoglobin  with  a  saturated  solution  of  potassium 
ferricyanide  until  the  color  has  changed  to  a  port-brown.  The 
mixture  is  cooled  to  0°  C,  and  treated  with  one-quarter  of  its 
volume  of  cooled  alcohol.  When  kept  in  the  refrigerator  crystal- 
lization takes  place  in  the  course  of  a  few  days.  The  crystals  are 
of  a  brown  color,  and  occur  as  needles,  prisms,  or  hexagonal  plate- 
lets. They  may  be  purified  by  recry stall ization  from  water  in  the 
presence  of  alcohol.  An  aqueous  solution  of  the  substance  is 
brown,  w^hile  its  alkaline  solutions  are  beautifully  red.  On  exposure 
to  sunlight  its  neutral  and  dilute  solutions  gradually  assume  a 
dark-red  color,  which  is  thought  to  be  referable  to  a  transformation 
of  methfemoglobin  into  photomethcemoglobin.  On  spectroscopic 
examination  such  solutions  show  one  broad  band  of  absorption  in 
the  green  portion  of  the  spectrum  no  matter  whether  the  reaction  is 
alkaline,  neutral,  or  acid.  Methaemoglobin  proper  under  the  same 
conditions  gives  a  fairly  broad  band  of  absorption  between  C  and  D, 
nearer  C,  w4iich  is  characteristic,  and  disappears  on  the  addition  of 
sodium  hydrate  solution.     In  addition,  two  different  bands  may  at 


362  THE  BLOOD. 

times  be  seen  between  D  and  E,  which  are  thought  to  be  referable, 
however,  to  a  contamination  of  the  substance  with  haemoglobin. 
Some  observers  further  speak  of  an  additional  band  near  F,  but 
this  is  not  characteristic.  On  reduction  of  the  alkalinized  solution 
with  ammonium  sulphide  the  spectrum  of  hamochromogen  results. 

According  to  v.  Zeynek,  photomethcemoglobhi  is  in  reality  cyan- 
haemoglobin,  and  is  formed  through  the  action  upon  the  haemoglobin 
of  hydrocyanic  acid  which  results  from  the  ferricyanide  in  conse- 
quence of  the  effect  of  sunlight.  Pure  methsemoglobin  is  not 
affected  by  sunlight. 

Like  oxy haemoglobin,  methsemoglobin  is  capable  of  combining 
with  certain  gases  to  form  molecular  compounds.  Of  these,  a 
carbon  dioxide  methcemoglobin,  a  methcemoglobin  sulphide,  and  a  cyan- 
methmmoglobin  have  been  described.  Acetylene  also  is  said  to  enter 
into  combination  with  the  coloring-matter  of  the  blood.  These 
compounds,  however,  are  but  little  known.  The  methaemoglobin 
sulphide  results  when  hydrogen  sulphide  and  air  are  simultaneously 
passed  through  lake-colored  blood.  It  gives  rise  to  a  greenish-red 
color,  and  it  is  thought  that  the  greenish  discoloration  of  decom- 
posing bodies  is  referable  to  its  presence.  On  spectroscopic  exami- 
nation its  neutral  solutions  give  two  bands  of  absorption  between 
C  and  D,  of  which  one  is"  brighter  and  located  near  C,  while  the 
other  and  darker  band  occupies  the  middle  portion  between  C  and 
D.  The  two  are  united  by  a  diffuse  shadow.  On  adding  a  strong 
solution  of  sodium  hydrate  the  darker  band  disappears,  and  if  now 
the  solution  is  heated  and  treated  with  a  reducing  agent,  the  spectrum 
of  hsemochromogen  results. 

The  substance  itself  has  not  been  isolated. 

Hsematoporphyrin. — This  substance,  as  has  been  indicated,  results 
from  hfematiu  when  this  is  treated  with  concentrated  sulphuric 
acid  that  has  been  saturated  with  hydrobromic  acid.  During  this 
process  the  iron  of  the  hamatin  is  split  off,  and  a  new  pigment, 
hoematoporphyrin,  is  formed.  In  the  circulating  blood  of  the  verte- 
brate animals  it  is  not  found  under  normal  conditions,  but  is  appar- 
ently formed  in  certain  diseases,  and  during  the  long-continued 
administration  of  sulphonal  and  related  bodies,  as  also  in  lead 
poisoning  and  following  intestinal  hemorrhages,  when  it  may  also  be 
found  in  the  urine.  Among  invertebrate  animals  it  is  said  to  occur 
in  the  integument  of  the  star-fish,  in  certain  snails,  in  the  earth- 
worm, in  various  sponges,  etc. 

Hsematoporphyrin  is  thought  to  be  isomeric  with  bilirubin,  and  is 
thus  represented  by  the  formula  C32H3gX^O,;.  On  reduction  it  yields 
a  pigment  which  is  apparently  identical  with  Maly's  hydrobilirubin, 
and  Jaffe's  urobilin.  It  may  be  obtained  in  crystalline  form  as 
a  hydrochlorate,  while  the  pigment  itself  is  amorphous.  Its  solu- 
tions in  acid  alcohol  present  a  l)eautiful  purple  color,  Avhich  is 
changed  to  a  violet  blue  on  adding  an  excess  of  the  acid.  It  is 
most  conveniently  obtained  by  starting  with  hremin  and  decomposing 


CHEMICAL  EXAMINATION  OF  THE  BLOOD.  363 

this  with  glacial  acetic  acid  that  has  been  saturated  with  hydro- 
bromlc  acid.  Its  solutions  in  acid  alcohol  give  two  bands  of  absorp- 
tion. One  of  these  is  located  between  C  and  D,  while  the  second 
band,  which  is  much  darker  and  more  strongly  defined,  occupies 
a  position  midway  between  D  and  E,  and  extends  as  a  shadow 
toward  D.  In  dilute  alkaline  solutions,  on  the  other  hand,  we  find 
four  bands :  one  between  C  and  D  ;  a  second  one,  which  is  broader 
than  the  first,  between  D  and  E  and  about  D ;  a  third  band, 
between  D  and  E,  near  E  ;  and  finally  a  further  band  between  b  and 
F,  which  is  the  widest  and  much  darker  than  the  rest.  On  treating 
with  an  alkaline  solution  of  zinc  chloride  this  spectrum  gradually 
passes  into  a  new  spectrum  with  only  two  bands,  of  which  one  is 
seen  about  D,  and  the  other  between  D  and  E. 

The  transformation  of  acid  ha^matoporphyrin  to  the  alkaline  form 
can  be  readily  effected  by  oversaturating  a  solution  of  htematopor- 
phyrin  in  sulphuric  acid  with  pyridin  ;  the  garnet  color  changes  to  a 
chestnut,  while  the  solution  remains  perfectly  clear. 

Several  anhydrides  of  h?ematoporphyrin  have  been  described. 
One  of  these,  C3„H34N^O-„  is  obtained  by  rubbing  up  hsemin  crystals 
with    concentrated    sulphuric    acid ;   another    has    the    composition 

C32H32Np^.    ^ 

On  oxidation  hpematoporphyrin  yields  the  same  ha^matinic  acids 
as  hfematin  (see  above).  The  resulting  tribasic  acid,  C^HyOg,  on 
reduction  with  hydriodic  acid  is  transformed  into  the-tribasic  hsemo- 
tricarbonic  acid,  CgHi20|; ;  this  is  ])0ssibly  identical  with  ethyl- 
triearballylic  acid,  which  has  been  obtained  synthetically. 

Closely  related  to  hrematoporphyrin,  apparently,  is  the  phyllopor- 
phyrin  which  may  be  obtained  from  the  chlorophyl  of  plants. 
From  its  formula,  C32ll34N^Oo,  and  that  of  hsematoporphyrin  anhy- 
dride, CgjHj^N^Oj,  it  is  suggested  that  both  are  different  oxidation- 
products  of  one  and  the  same  substance.  On  reduction  with  phos- 
phonium  iodide  in  glacial  acetic  acid  hfematoporphyrin  yields  a 
product  of  the  composition  C34H.^OgN4,  which  Xencki  and  Saleski 
designate  as  mesoporphyrin.  This  manifestly  occupies  a  position 
intermediate  between  hsematoporphyrin  and  phylloporphyrin.  On 
still  further  reduction  methyl-propyl-pyrrol  is  obtained  (see  Hsema- 
tin),  and  it  is  noteworthy  that  the  same  body  has  also  been  obtained 
in  the  same  manner  from  phylloporphyrin.  AVe  see  that  the  prin- 
cipal animal  pigment  is  thus  intimately  related  to  the  principal  pig- 
ment of  the  higher  plants.  The  spectra  of  haematoporphyrln,  meso- 
porphyrin,  and  phylloporphyrin  are  very  similar ;  they  differ  from 
one  another  by  only  a  slight  displacement  of  all  bands  in  one  direction. 

Haematoidin. — This  pigment,  which  was  first  observed  by  Vir- 
chow  in  old  extravasations  of  l)lood,  in  which  it  may  occur  in  crystal- 
line form,  is  now  known  to  be  identical  with  bilirubin.  As  a  separate 
substance  it  therefore  no  longer  merits  consideration.  Its  develop- 
ment from  blood-pigment,  however,  demonstrates  the  close  relation 
existing  between  it  and  the  coloring-matter  of  the  bile  (which  see). 


364  THE  BLOOD. 

I  have  pointed  oiir  that  in  some  of  the  lower  animals  haemoglobin 
is  also  found,  and  may  occur  in  the  blood  either  as  such  or  bound  to 
certain  cellular  elements  which  may  be  compared  to  the  red  cor- 
puscles of  the  vertebrates.  In  other  invertebrate  animals  we  find 
no  haemoglobin,  but  related  respiratory  pigments,  which  are  partly 
violet  or  purplish  red  in  color,  and  partly  blue.  The  former  com- 
prise the  so-called  ^on'cZms,  of  which  little  is  known,  while  the  latter 
group  is  represented  by  the  oxy-compound  of  hsemocyanin. 

Hsemocyanin  is  of  special  interest,  as  it  is  ai)})ai-ently  closely 
related  to  haemoglobin,  but  contains  copper  in  its  molecule  in  the 
place  of  iron.  Unlike  haemoglobin,  however,  haemocyanin  is  itself 
colorless,  while  its  oxy-compound,  oxyhaemocyanin,  presents  a  beau- 
tiful blue  color.  According  to  Frederique,  oxyhsemocyanin  yields 
an  albuminous  substance  on  decomposition  with  hydrochloric  acid  as 
also  with  nitric  acid,  which  may  be  compared  to  globin,  and  a  cop- 
per-containing pigment  which  corresponds  to  luematin.  Heuzel 
however,  was  unable  to  confirm  this  observation.  He  obtained  an 
albuminous  body  of  the  character  of  an  acid  albumin,  but  no  histon. 
The  copper  of  the  hremocyanin  is  not  present  in  firm  organic  combi- 
nation, but  in  a  loosely  combined  form,  analogous  to  a  copper  albu- 
minate. 

Henze  has  recently  succeeded  in  crystallizing  the  hsemocyanin,  and 
on  elementary  analysis  obtained  the  following  values  :  C  ^^  53.66  ; 
H  =  7.33  ;  N  =  16.09  ;  S  =  0.86  ;  Cu  =  0.38,  and  O  =  21.67. 

On  reduction  with  ammonium  sulphide  or  on  exposure  to  an  at- 
mosphere of  an  indifferent  gas  like  H,  COo,  N,  etc.,  the  oxyhsemo- 
cyanin yields  the  colorless  haemocyanin.  On  spectroscopic  examina- 
tion hsemocyanin  and  oxyhaemocyauin  show  a  shadow  at  both  ends 
of  the  spectrum,  which  is  more  marked  in  the  latter ;  true  absorption- 
bands,  however,  are  not  observed. 

Other  invertebrate  animals  contain  only  lipochromic  pigments  in 
their  haemolymph,  which  probably  do  not  possess  a  respiratory  func- 
tion however,  and  in  the  lowest  forms  of  life,  of  course,  special 
oxygen-carriers  are  not  required. 


CHAPTER    XV. 

THE  LYMPH. 

In  its  course  through  the  blood-capillaries  a  portion  of  the  plasma 
passes  out  through  the  vessel-walls  and  enters  a  system  of  irregular 
interfascicular  clefts,  which  are  bounded  by  bundles  of  fibrous  tissue 
and  constitute  the  radicles  of  the  lymphatic  system.  Through  these 
clefts  the  plasma  reaches  the  individual  cells  of  the  various  tissues 
and  organs  of  the  body,  and  supplies  these  with  the  requisite 
nourishment,  while  at  the  same  time  it  takes  up  the  waste  matter 
that  is  formed  in  the  metabolism  of  the  cells,  and  through  the 
lymph-vessels  carries  these  into  the  venous  current  of  the  blood. 
This  fluid,  which  thus  contains  the  various  constituents  of  the 
blood-plasma  and  the  decomposition^products  of  the  cells,  is  termed 
the  lymph. 

In  addition  to  the  lymph-vessels  proper  and  their  radicles,  the 
lymph-clefts,  this  fluid  is  also  found  in  the  so-called  serous  cavities 
of  the  body,  viz.,  the  pleura,  the  peritoneal  and  pericardial  cavities, 
in  the  ventricles  of  the  brain  and  the  spinal  cord,  in  the  communi- 
cating subarachnoid  space,  and  also  in  the  anterior  chamber  of  the 
eye.  In  health,  however,  these  cavities  contain  but  little  fluid,  and 
quantities  sufficient  for  analytical  purposes  can  normally  be  obtained 
only  from  the  pericardial  sac,  and  at  times  from  the  subarachnoid 
space.  Under  pathological  conditions,  however,  large  accumulations 
of  fluid  may  be  observed,  and  not  only  in  the  serous  cavities  of  the 
body,  but  also  in  the  areolar  connective  tissue,  beneath  the  skin, 
and  beneath  the  muscles.  When  due  to  circulatory  disturbances,  a 
hydrsemic  condition  of  the  blood,  or  an  insufficient  elimination  of 
water  through  the  kidneys,  silch  accumulations  of  fluid  are  spoken 
of  as  transudates,  while  the  term  exudates  is  applied  to  similar 
accumulations  of  inflammatory  origin. 

Formerly  it  was  supposed  that  the  lymph  resulted  from  the  blood- 
plasma  through  a  simple  process  of  filtration  or  transudation  only, 
and  in  accordance  with  this  view  we  find  that  in  the  various  accumu- 
tions  of  lymph  the  salts  and  extractives  are  present  in  about  the 
same  amount  as  in  the  blood-plasma.  Heidenhain,  however,  has 
shown  that  the  flow  of  the  lymph-current  is  far  too  sluggish  to 
supply  the  various  organs  of  the  body  with  the  proper  amount  of 
nourishment,  supposing  its  composition  to  be  everywhere  the  same 
as  that  of  the  blood-plasma.  We  are  hence  forced  to  the  conclusion 
that  the  endothelial  cells  of  the  capillaries  possess  a  selective  secre- 
tory power  similar  to  that  of  the  renal  epithelium,  and  are  thus 

365 


366  THE  LYMPH. 

capable  of  furnishing  to  each  tissue  its  proper  amount  and  kind  of 
food.  This  view,  however,  does  not  preclude  the  possibility  that 
some  of  the  constituents  of  the  plasma  may  pass  over  into  the  lymph 
by  a  simple  process  of  filtration,  and  it  is  likely  that  this  actually 
occurs  with  the  water  and  most  of  the  mineral  salts. 

According  to  its  origin,  then,  we  may  expect  to  find  certain  dif- 
ferences in  the  chemical  composition  of  the  lymph,  and  w^e  find,  as 
a  matter  of  fact,  that  such  differences  exist.  These  are,  however, 
essentially  of  a  quantitative  kind,  and  qualitatively  we  find  the  same 
constituents  in  the  lymph  from  the  various  districts  as  compared  with 
each  other  and  with  the  blood-plasma. 

Like  the  blood-plasma,  the  lymph  consists  of  a  liquid  portion,  the 
lymph-plasma,  and  cellular  elements,  the  lymph-corpuscles.  These 
latter  are  essentially  mononuclear  leucocytes,  and  are  largely  derived 
from  the  lymphoid  tissue  which  abounds  in  the  course  of  the  lymph- 
vessels.  Red  corpuscles  are  either  lacking  entirely  or  they  are  pre- 
sent in  very  small  numbers.  They  are  of  much  darker  color  than 
those  of  the  blood,  but  on  exposure  to  the  air  they  take  on  the 
bright-red  color  of  oxy haemoglobin.  According  to  some  observers, 
they  represent  transition-forms  between  the  leucocytes  and  the 
normal  red  corpuscles  of  the  blood. 

In  various  inflammatory  diseases  of  the  serous  cavities  the  leuco- 
cytes may  be  present  in  very  large  numbers,  and  in  extreme  cases, 
indeed,  they  predominate  to  such  an  extent  that  the  liquid  character 
of  the  lymph  may  be  almost  entirely  lost. 

The  appearance  of  the  lymph  is  dependent  upon  the  number  of 
the  leucocytes  and  the  amount  of  fat  present.  As  obtained  from 
fasting  animals,  it  represents  a  slightly  viscid,  straw-  or  rose-colored, 
transparent  fluid.  During  the  process  of  digestion,  on  the  other 
hand,  and  especially  after  the  ingestion  of  much  fatty  food,  it 
becomes  more  or  less  opaque,  owing  to  the  admixture  of  the  fat, 
which  is  carried  into  the  general  lymph-current  through  the  chyle, 
viz.,  the  lymph  coming  from  the  intestinal  canal. 

The  odor  of  the  lymph,  like  that  of  the  blood,  is  different  in 
different  animals.  Its  taste  is  salty,  and  the  reaction  slightly  alka- 
line.    The  specific  gravity  varies  between  1.015  and  1.021. 

The  amount  of  lymph  w^hich  is  produced  in  the  twenty-four  hours 
is  largely  influenced  bv  the  process  of  digestion.  During  starvation 
a  smaller  amount  is  thus  found  than  after  the  ingestion  of  food,  and 
it  appears,  moreover,  that  an  albuminous  diet  causes  a  much  greater 
increase  than  one  of  carbohydrates.  Active  nmscular  exercise  has 
a  similar  stimulating  effect  upon  its  formation.  Artificially  the 
amount  of  lymph  can  be  increased  by  the  intravenous  injection  of 
so-called  I//mphagogues,  of  which  Heidenhain  recognizes  two  classes, 
viz.,  those  which  merely  increase  the  amount  of  water  in  the  lymph 
and  those  which  also  luring  about  an  increase  of  the  organic  solids. 
The  former  include  such  crystalline  substances  like  sugar,  urea, 
sodium  chloride,  etc, ;  and  Heidenhain  supposes  that  their  action  is 
dependent  upon  their  passage  into  the   lymph,   where  they  exert 


THE  LYMPH.  367 

a  stimulating  effect  upon  the  cells  of  the  tissues  and  cause  an 
absorption  of  cellular  water.  The  latter,  on  the  other  hand,  are  in 
part  unknown  substances  which  can  be  extracted  from  the  muscles 
of  the  crab,  from  the  head  and  body  of  leeches,  from  the  bodies  of 
anodonts,  from  the  liver  and  intestines  of  the  dog,  and  also  com- 
prise the  peptones  and  egg-albumin.  The  influence  of  these  sub- 
stances is  apparently  exerted  upon  the  endothelial  cells  of  the 
blood-capillaries,  whereby  the  secretory  power  is  increased,  and 
we  accordingly  find  more  albumin  in  the  lymph  than  in  the  remain- 
ing blood-plasma.  Such  lymph  then,  in  contradistinction  to  the 
cellular  lymph  which  is  found  in  the  first  instance,  may  be  termed 
blood-lymph.  The  state  of  the  blood-pressure,  according  to  Heiden- 
hain,  is  of  no  moment  in  bringing  about  these  changes,  and  he  found, 
as  a  matter  of  fact,  that  variations  between  10  and  20  Hgmm.  on  the 
one  hand,  and  150  to  200  Hgmm.  on  the  other  hand,  are  of  little 
influence  upon  the  amount  of  lymph  that  is  produced.  This  view, 
however,  is  in  all  probability  not  final. 

According  to  Bunge,  the  amount  of  lymph  that  is  formed  in  the 
twenty-four  hours  by  the  human  being  amounts  to  about  4000  c.c. 

A  general  idea  of  the  chemical  composition  of  lymph  may  be 
formed  from  the  following  analysis  of  Munck  and  Rosenstein. 
The  material  was  obtained  from  a  fistula  in  the  thigh  of  a  young 
woman.  Accompanying  this  is  an  analysis  of  the  blood-plasma 
(taken  from  Hammarsten)  for  comparison  : 

Lymph.  Blood-plasma. 

Water 96.5     -94.5  per  cent.  91.8  per  cent. 

Solids 3.7     -5.5  "  "  8.2    "      " 

Albumins 3.4     -4.1  "  "  6.9    "      " 

Ethereal  extract 0.06  -0.13  "  " 

Sugar       0.1  "  "  0.46  "      " 

Salts 0.8     -0.9  "  "  0.84"      " 

Sodium  chloride    .    .    .  0.55  -0.58  "  " 

Sodium  carbonate      .    .     0.24  "  " 

Disodic  phosphate     .    .  0.028  "  " 

Of  these  constituents,  the  fat  is  subject  to  the  greatest  variations, 
and  is,  of  course,  always  more  abundant  during  the  process  of  diges- 
tion in  the  chyle  than  in  any  other  lymphatic  district.  In  Munck's 
case  the  amount  rose  to  4.7  per  cent,  after  the  ingestion  of  a  large 
amount  of  fatty  food,  and  decreased  to  0.06-0.26  per  cent,  when 
food  was  withheld  for  twenty-four  hours. 

The  flxt  is  present  in  the  lymph  to  the  greatest  extent  as  neutral 
fat,  and  it  is  to  be  noted  that  it  here  exists  in  a  state  of  emulsion,  so 
that  upon  microscopical  examination  the  chyle  more  especially  will 
be  seen  to  contain  innumerable  fat  droplets,  which  vary  but  little  in 
size  and  have  no  tendency  to  flow  together,  as  in  the  case  of  milk. 
Why  this  is  we  do  not  know,  but  it  has  been  suggested  that  each 
fat  droplet  is  surrounded  by  a  delicate  albuminous  envelope,  which 
is  derived  from  the  normal  albumins  of  the  lymph.  On  the  other 
hand,  it  is  conceivable  that  the  surface  layer  of  each  droplet  con- 


368  THE  LYMPH. 

sists  of  modified  fat  or  of  a  denser  layer  of  the  fluid  in  which  it  is 
suspended. 

The  fats  which  are  found  in  the  lymph  are  always  identical  M'ith 
those  of  the  food,  and  can  be  recovered  for  analytical  purposes  by 
extracting  with  ether.  In  its  course  through  tissues  which  are  rich 
in  fat  no  fat  is  absorbed. 

Soaps  are  present  in  the  lymph  in  only  very  small  amounts. 

The  albumins  which  are  found  in  the  lymph  are  the  same  as  those 
of  the  blood,  and  are  present  in  the  same  ratio  to  each  other.  The 
amount  varies  somewhat  with  the  character  of  the  lymph,  Ijut  is 
normally  always  smaller  than  that  of  the  blood-plasma.  Like  the 
blood-plasma,  so  also  does  the  lymph  coagulate  on  standing.  The 
coagula,  however,  are  very  delicate  and  tend  to  separate  out  in  frac- 
tions. Some  transudates,  indeed,  such  as  pericardial  effusions  and 
hydrocele  fluid,  do  not  coagulate  spontaneously  at  all,  while  coagula- 
tion occurs  at  once  if  leucocytes  or  blood  is  added.  The  peculiar 
behavior  of  such  lymph  is  no  doubt  due  to  the  absence  of  cellular 
elements.  It  is  to  be  noted  also  that  following  the  injection  of 
those  substances  which  prevent  coagulation  of  the  blood  coagula- 
tion of  the  lymph  similarly  does  not  occur. 

Other  all)umins  besides  serum-albumin,  serum -globulin,  and  fibrin- 
ogen are  not  found  in  normal  lymph,  such  as  we  obtain  from  the 
thoracic  duct,  for  example.  Under  pathological  conditions,  how- 
ever, the  exudates  more  particularly  may  contain  the  various  albu- 
minous derivatives  of  the  leucocytes,  such  as  nucleins,  nucleo- 
albumins,  etc.  In  cysts  of  the  ovaries  and  their  appendages  met- 
alhimin  or  paraJbu7nin  may  further  be  found. 

The  amount  of  sugar  which  is  found  in  normal  lymph  is  fairly 
constant,  and  is  derived  from  the  hepatic  lymph.  It  can  be  increased 
artificially  by  ligating  the  ureters  and  then  injecting  glucose  into 
the  blood.  It  is  noteworthy  that  under  such  conditions  the  lymph 
mav  contain  a  larger  percentage  of  sugar  than  the  blood  itself.  This 
further  shows  that  the  formation  of  lymph  cannot  be  explained 
upon  the  basis  of  filtration  and  osmosis  only,  and  demonstrates 
the  specific  activity  of  the  endothelial  lining  of  the  capillaries. 
Under  pathological  conditions,  it  is  claimed,  sugar  may  be  altogether 
absent. 

The  extractives  of  the  lymph  are  essentially  the  same  as  those  of 
the  plasma.  In  certain  districts,  however,  the  one  or  the  other  will 
be  found  to  preponderate,  and  in  some  localities  we  further  meet 
with  extractives  which  are  peculiar  to  that  particular  region.  In 
the  cerebrospinal  fluid,  for  example,  pyrocatechin  has  been  found  ; 
allantoin  is  present  in  the  allantoic  fluid  and  in  ascitic  accumula- 
tions ;  succinic  acid  and  inosit  may  be  obtained  from  hydrocele 
fluid  in  which  cholesterin  may  also  be  present  in  very  considera]>le 
amount.  Of  interest  also  is  the  fact  that  in  the  lymph  of  the 
thoracic  duct  bile  acids  can  be  demonstrated,  and  it  appears  from  the 
researches  of  Croftan  that  the  acids  in  question  are  present  in  the 
leucocytes.     They  supposedly  represent  that  portion  of  the  bile  acids 


THE  LYMPH.  369 

which  has  escaped  decomposition  in  the  intestinal  canal  and  has 
been  resorbed.  They  subsequently  enter  the  blood-current,  still 
held  by  the  leucocytes,  and  are  again  eliminated  in  the  bile. 

Traces  of  urea,  uric  acid,  lecithin,  xanthin,  kreatin,  and  lactic 
acid  are  commonly  found. 

Of  ferments,  we  find  a  diastatic  ferment  and  the  glucolytic  fer- 
ment of  Lepine. 

The  gases  of  the  lymph  differ  from  those  of  the  blood  in  the 
presence  of  larger  amounts  of  carbon  dioxide — 35  to  45  per  cent. — 
as  compared  with  arterial  blood,  and  smaller  amounts  than  are 
found  in  venous  blood.  The  tension  of  the  carbon  dioxide,  as 
compared  with  venous  blood,  is  lower,  which  suggests  that  a  por- 
tion of  the  gas  enters  the  blood  from  the  lymph  through  a  reverse 
secretory  activity  of  the  capillary  endothelium.  Oxygen  is  present 
only  in  traces,  while  the  amount  of  nitrogen  is  the  same  in  both,  viz., 
1.6  per  cent. 

For  purposes  of  comparison  and  reference,  a  few  analyses  of  some 
of  the  more  important  normal  and  pathological  varieties  of  lymph 
are  appended.  Some  of  these  will  be  considered  in  greater  detail 
in  subsequent  chapters. 

Analysis  op  Human  Peeicardial  Fluid  (Hammarsten). 

Water 960.85 

Solids 39.14 

Albumins 28.60 

Fibrin 0.31 

Globulins 5.95 

Serum-albumin 22.34 

Extractives       2.00 

Soluble  salts 8.60 

Sodium  cliloride 7.28 

Insoluble  salts 0.15 


Analysis  of  Dog's  Chyle  (Hoppe-Seyler). 

Water 906.77 

Solids 96.23 

Fibrin 1.11 

Albumins  and  globulins 21.05 

Fats  ) 

Lecithin        V 64.86 

Cholesterin  J 

Fatty  acids  and  soaps  \  2  04 

Other  organic  bodies  J       

Mineral  salts 7.92 


Analysis  op  Aqueous  Humor  of  Calf  (Halliburton). 

Water 986.87 

Solids    .    .  _ 13.13 

Albumins 1.12 

Extractives .  4.21 

Inorganic  salts 7.70 

Sodium  chloride 6.89 

24 


370  THE  LYMPH. 

Analysis  of  Cerebrospinal  Fluid  (Gautier). 

Water 987.00 

yolids 10.59 

Albumins 1.10 

Fats 0.09 

Cliolesterin .    .    .    .      0.21 

Alcoholic  and  aqueous  extracts,  minus  salts,  but  including 

sodium  lactate 2.75 

Salts      6.44 

Chlorides 6.14 

Sulphates      0.20 

Earthy  phosphates      0.10 

Ammonia traces. 

Analyses  of  Pleural  Effusions  (Gautier). 

Acute 
pleurisy. 

Water 937.60 

Organic  matter 54.40 

Fibrin 0.09 

Mineral  matter 8.00 


Chronic 

Hvdrothorax 

pleurisy. 

^cardiacj. 

933.80 

958.70 

58.20 

32.30 

0.00 

0.19 

8.00 

9.00 

>rivon  and  Scherer). 

Chronic 

Hepatic 

nephritis. 

cirrhosis. 

978.00 

984.50 

Ovarian 
cancer. 

Water 946.50 

Serum-albumin 19.40 

Serum-globulin      18.58 1                 g^Q                    g  j- 

Mucm(?j 0.9oJ 

Fats              )  1  q    -> 

Cholesterin   V 1.25                    gQO  I                 '^'-^ 

Extractives  J 

Soluble  salts 5.5^                 s.OO                    8.46 

Insoluble  salts /  .oo  j 

Analysis  of  Hydrocele  Fluid  (Hammarsten). 

Water 938.85 

SoUds 61.15 

Fibrin 0.59 

Globulins      13.25 

Serum-albumin 35.94 

Ethereal  extract 4.02 

Soluble  salts 8.60 

Insoluble  salts 0.66 

Analysis  of  Amniotic  Fluid  (Labroche). 

Human. 

Water 987.300 

Solids 16.700 

Serum-albumin 2.590 

Mucin         1 

Albumoses  [■ 1.604 

Glucose      J 

Urea      0.450 

Fats 0.356 

Mineral  salts 7.695 

Sodium  chloride 6.071 

Disodium  phosphate 1.621 

Sulphates traces. 

Salts  of  calcium  and  potassium none. 


THE  LYMPH.  371 

Analysis  of  Lymph  from  Cellular  Tissue  ((Edema). 

Water 975.20 

Albumins 5.42 

Fats  and  extractives 3.76 

Mineral  salts 15.62 

Analysis  of  Pus. — The  composition  of  the  leucocytes  which 
enter  into  the  formation  of  pus  has  been  considered  (pages  324 
and  350).  An  analysis  of  pus-serum  is  here  given,  which  is  taken 
from  Robin  : 

Water 937.90 

Metalbumin        '\ 

Serum-albumin  >■ 11.00 

Serum-globulin  J 

Lecithin 6.00 

Fats  and  soaps 10.00 

Cholesterin      3.50 

Serolin      1.00 

Leucin,  tyrosin,  and  extractives  in  general 15.00 

Salts  of  organic  acids traces. 

Sodium  chloride      3.11 

Sodium  phosphate  . traces. 

Phosphates  of  calcium  and  magnesium 0.50 

Sulphates 1.87 

Salts  of  iron  and  silica      0. 16 

THE  SYNOVIAL  FLUID. 

The  synovial  fluid,  though  not  a  lymph  in  the  narrower  sense  of 
the  term,  is  for  convenience'  sake  briefly  described  at  this  place.  It 
is  the  specific  secretion  of  the  synovial  membrane  of  the  joints, 
and  constitutes  a  strongly  alkaline,  viscid,  yellowish,  somewhat 
cloudy  fluid.  In  addition  to  the  common  albumins,  fats,  salts,  and 
extractives  of  the  lymph,  it  contains  also  a  peculiar  mucinous  body, 
which  is  termed  synovia,  and  apparently  belongs  neither  to  the 
nucleo-albumins  nor  the  mucins  or  mucoids.  It  can  be  precipitated 
with  acetic  acid  and  coagulates  on  the  application  of  heat.  Of  its 
nature,  however,  nothing  further  is  known.  In  addition,  another 
mucin-like  body  is  found  which  is  rich  in  phosphorus,  and  probably 
belongs  to  the  nucleo-albumins. 

Quantitatively  the  composition  of  the  synovial  fluid  varies  with 
exercise  and  rest  in  such  a  manner  that  on  motion  the  mucinous 
body,  as  also  the  albumins  and  extractives,  increase,  while  the  salts 
diminish.  This  is  shown  in  the  appended  analyses,  which  are  taken 
from   Frerichs : 

stalled  ox.  p0^t°^^ 

Water 969.90  948.54 

Solids 30.10  51.50 

Mucin  (?)      2.40  5.60 

Albumins  and  extractives 15.76  35.12 

Fats 0.62  0.76 

Salts 11.32  9.98 


CHAPTEK    XVI. 

THE  MUSCLE-TISSUE. 

I  HAVE  pointed  out  that  while  in  the  monocellular  organisms  the 
various  functions  of  the  body  are  carried  on  by  the  single  cell,  a 
gradual  division  of  labor  occurs  as  we  ascend  in  the  scale  of  both 
animal  and  vegetable  life,  where  groups  of  cells  are  set  aside  for  the 
performance  of  certain  special  functions.  Structurally  this  division 
of  labor  finds  its  expression  in  a  more  or  less  well-marked  deviation 
from  the  original  type,  as  has  been  shown.  At  first  sight,  it  is 
thus  difficult  to  connect  the  highly  differentiated  muscle-cell  with 
the  apparently  much  more  simple  ovum  from  which  it  has  originated. 
The  element  of  reproduction  and  secretion  is  here  manifestly  placed 
in  the  background,  while  in  its  co-ordinate  and  rapid  contraction  on 
stimulation  we  have  abundant  evidence  of  its  highly  specialized 
function.  That  this  should  further  be  expressed  in  the  chemical 
composition  of  the  cells  suggests  itself  at  once.  As  a  matter  of 
fact,  we  here  find  substances  which  may  be  regarded  as  specific 
muscle  components,  and  it  seems  warrantable  to  assume  that  a 
definite  connection  exists  between  these  bodies  and  the  special  func- 
tion of  the  cell.  On  chemical  examination  we  may  then  further 
expect  to  meet  with  the  various  products  of  katabolism,  so  far  as 
these  are  found  in  the  muscle-tissue  proper,  and  have  not  as  yet  been 
removed  by  the  blood  or  the  lymph. 

Before  proceeding  to  a  study  of  these  various  substances  in  detail, 
a  few  analyses  of  muscle-tissue  are  here  introduced,  which  will 
furnish  a  general  idea  of  its  chemical  composition.  Qualitatively 
this  is  fairly  constant,  but  quantitative  variations  occur  which  are 
often  very  marked. 

In  preparing  the  tissue  for  analytical  purposes,  the  blood  should 
first  be  washed  out  entirely  with  dilute  saline  solution  (0.6  per  cent.). 
Fibrous  tissue  and  fat  must  be  dissected  away  as  far  as  possible  and 
all  larger  bloodvessels  removed.  The  material  is  then  further 
scraped,  so  as  to  get  rid  of  as  much  of  the  connective  tissue  as  pos- 
sible which  binds  the  individual  fibres  together,  and  is  now  ready 
for  examination. 

Analyses  of  Fresh  Muscle-tissue  (Neumeister). — The  figures 
represent  average  values,  which  have  been  collected  from  various 
sources,  and  have  reference  to  mammalian  muscle-tissue  in  general, 
unless  otherwise  stated. 

372 


THE  MUSCLE-ALBUMINS.  373 

Per  cent. 

Water 75.50 

Solids 24.50 

Organic  constituents 23.50 

Myosin  (?) 7.74* 

Nucleins 0.37 

Albumins,  proteids,  and  albuminoids  (insoluble  in 

neutral  solution) 15.25 

Collagen   (referable  to  interfibrillary   connective 

tissue) 3.16 

Fats 3.71 

Glycogen 0.7-1.0 

Lactic  acid 0.1-1.0 

Inosit 0.003 

Kreatin 0.21-0.28 

Xanthin 0.01-0.1^ 

Hypoxanthin 0.04-0.12 

Guanin      0.005 

Inorganic  constituents 1.000 

Phosphoric  acid 0.4674 

Chlorine 0.0672 

Potassium  oxide 0.4654 

Sodium  oxide 0.0770 

Calcium  oxide 0.0086 

Magnesium  oxide 0.0412 

Iron  oxide 0.0057 

In  studying  this  analysis  we  observe  that,  aside  from  the  mineral 
constituents,  various  bodies  are  encountered  here  which  represent 
distinct  products  of  katabolism,  and  which  are  hence  most  likely  not 
concerned  in  the  specific  function  of  the  muscle-tissue.  These  com- 
prise the  common  extractives,  viz.,  xanthin,  hypoxanthin,  guanin, 
kreatin,  lactic  acid,  and  possibly  also  inosit.  They  are  no  doubt 
formed  as  a  consequence  of  cellular  activity,  but  play  no  role  in  the 
function  of  the  muscle  proper.  On  the  other  hand,  we  meet  with 
food-stuifs  proper,  viz.,  albumins,  carbohydrates,  and  fats,  and  we 
mav  a  priori  expect  that  all  these  substances,  conjointly  or  in- 
dividually, are  directly  concerned  in  the  contractile  function  of 
the  cell. 

THE   MUSCLE-ALBUMINS. 

As  in  the  case  of  all  tissues  of  the  body,  the  muscle-tissue  also 
consists  of  a  liquid  portion,  the  so-called  muscle-plasma,  and  a  more 
solid  portion,  which  may  be  termed  the  muscle-sfroma. 

Muscle -plasma  may  be  obtained  in  the  following  manner  :  while 
the  animal  is  still  livino;  the  blood  is  thoroughly  washed  from  the 
large  skeletal  muscles  by  injecting  into  the  larger  arteries  a  dilute 
saline  solution  that  has  been  warmed  to  the  temperature  of  the  body, 
and  allowing  the  fluid  to  escape  from  the  corresponding  veins.  This 
is  continued  after  death  until  the  outflowing  water  is  colorless.  The 
muscles  are  then  rapidly  dissected  off,  ground  to  a  pulp  together 
with  pumice-stone,  and  passed  through  a  filter-press.  The  resulting 
liquid  is  the  muscle-plasma. 

1  In  birds 


374  THE  MUSCLE-TISSUE. 

While  this  procedure  is  applicable  in  the  case  of  mammalian 
muscle-tissue  in  general,  special  precautions  are  necessary  if  the 
muscles  of  the  lower  animals  are  to  be  studied.  In  the  frog,  for 
example,  the  tissue,  after  removal  of  the  blood,  must  be  frozen  in 
a  gradual  manner,  after  which  the  entire  process  is  continued  at 
a  temperature  below  — 3°  C.  A  snow-like  mass  is  finally  obtained, 
which  melts  at  — 3°  C. 

The  color  of  the  muscle-plasma  varies  with  the  color  of  the 
muscles  from  which  it  has  been  obtained.  In  the  case  of  the  dog  it 
is  of  a  brownish  color,  in  rabbits  it  is  yellowish  red,  and  in  frogs 
a  light  yellow.  The  particular  shade  of  color,  as  ^vill  be  seen  later, 
is  a  direct  expression  of  the  degree  of  functional  activity  of  the 
individual  muscles,  and  is  in  part  due  to  hsemoglobin  and  in  part  to 
certain  lipochromes. 

The  reaction  of  the  plasma  is  neutral  or  slightly  alkaline. 

On  standing  for  a  length  of  time  mammalian  muscle-plasma 
gradually  undergoes  a  process  of  coagulation,  but  it  is  to  be  noted 
that  the  coagulum  which  separates  out  is  slight  in  amount.  In 
the  case  of  the  frog,  on  the  other  hand,  the  entire  bulk  of  the 
plasma  becomes  gelatinous,  and,  in  contradistinction  to  the  mam- 
malian plasma,  this  process  begins  at  a  temperature  of  0°  C.  As  in 
the  case  of  the  blood-plasma,  the  coagulum  gradually  contracts,  and 
the  liquid  w' hich  remains  is  then  spoken  of  as  muscle-serum.  The  reac- 
tion is  then  acid.  The  substance  wdiich  composes  the  clot  is  termed 
inyog en-fibrin.  The  behavior  of  muscle-plasma  is  thus  quite  simi- 
lar to  that  of  blood-plasma,  and  here,  as  there,  the  resulting  fibrin 
is  derived  from  an  albuminous  substance  which  was  previously  pre- 
sent in  solution.  This  substance  is  here  termed  myogen.  In  addi- 
tion the  muscle-plasma  contains  another  albuminous  body,  myosin  ; 
and  it  appears  from  the  researches  of  v.  Fiirth  that  these  two  sub- 
stances are  the  only  soluble  albumins  Avhich  are,  contained  in  muscle- 
tissue,  if  we  disregard  a  variable  amount  of  a  soluble  myogen-fibrin, 
which  is  itself  a  derivative   of  myogen. 

Myogen. — Isolation. — Myogen  is  most  conveniently  obtained 
from  muscle-plasma  after  the  myosin  has  been  removed  by  the 
previous  addition  of  ammonium  sulphate  to  the  extent  of  28  per 
cent.  The  resulting  precipitate  is  removed  and  the  filtrate  saturated 
with  the  same  salt  in  substance.  The  myogen  is  thus  thrown  down 
together  with  the  soluble  myogen-fibrin.  It  is  washed  with  a  satu- 
rated solution  of  ammonium  sulphate,  dissolved  in  water,  and  freed 
from  the  soluble  myogen-fibrin  by  heating  to  40°  C,  when  this  is 
transformed  into  the  insoluble  form  and  is  filtered  oif.  The  remain- 
ing solution  contains  the  myogen  in  pure  form. 

In  its  general  pnjpcrties  it  resembles  the  albumins  proper,  in  con- 
tradistinction to  the  globulins.  It  is  soluble  in  water,  and  can  be 
precipitated  by  alcohol,  by  salting  with  ammonium  sulphate,  sodium 
cidoride,  and  magnesium  sulphate.  The  two  latter  salts,  however, 
do   not   cause   a   complete   precipitation.     Alcohol   (92   per  cent.) 


THE  MUSCLE-ALBUMINS.  375 

renders  the  substance  insoluble  to  a  slight  extent,  but  the  greater 
portion  is  refractory  in  this  respect. 

By  acetic  acid  myogen  is  precipitated  only  in  the  presence  of  a 
neutral  salt,  but  redissolves  in  an  excess  of  the  acid,  with  the  forma- 
tion of  syntonin.  The  tendency  to  the  transformation  into  albu- 
minates is  indeed  more  marked  in  the  case  of  the  soluble  muscle- 
albumins,  in  general,  than  with  any  other  forms.  Mineral  acids  are 
in  this  respect  still  more  active  than  acetic  acid,  and  as  a  conse- 
quence a  precipitation  of  myogen  is  observed  only  when  such  acids 
are  present  in  certain  proportion. 

Carbonic  acid  and  the  salts  of  the  heavy  metals  precipitate  myo- 
gen only  in  the  presence  of  a  neutral  salt. 

Myogen  coagulates  at  a  temperature  of  from  55°  to  65°  C.  It  is 
not  precipitated  on  dialysis. 

Elementary  analysis  of  a  myogen  preparation  obtained  from  the 
muscles  of  a  rabbit  2:ave  the  following  average  results  :  C  =  52.69  ; 
H  =  6.93  ;  N  =  16.20. 

When  solutions  of  myogen  are  kept  at  a  certain  temperature,  and 
in  the  presence  of  a  definite  amount  of  a  neutral  salt,  the  substance 
is  gradually  transformed  into  a  soluble  form  of  myogen-fibrin,  which 
differs  from  myogen  in  the  fact  that  it  is  thrown  down  on  dialysis, 
and  in  its  point  of  coagulation,  which  lies  at  40°  C.  As  has  been 
stated,  a  certain  amount  of  soluble  viyogen-Jihria  seems  to  occur  pre- 
formed in  the  muscle-tissue,  and  separates  out  gradually  on  standing. 
At  40°  C,  however,  this  occurs  instantaneously.  By  coagulation 
the  soluble  myogen-fibrin  is  transformed  into  the  insoluble  form,  the 
myogeii-fihrin  proper. 

The  relative  amount  of  myogen,  as  compared  with  myosin  (see 
belo-w),  which  is  found  in  muscle-tissue  varies  in  all  ^probability  with 
different  animals.  In  rabbits,  v.  Fiirth  observed  that  myogen  repre- 
sented about  80  per  cent,  of  the  total  amount  of  soluble  albumins. 

The  amount  of  soluble  myogen-fibrin  which  is  included  in  the 
above  figures  is  in  mammals  apparently  very  small,  as  only  slight 
coagula  are  formed  when  the  plasma  is  heated  to  40°  C.  But  in  the 
frog  large  amounts  are  manifestly  present.  In  some  animals,  on  the 
other  hand,  it  is  apparently  absent. 

Myosin. — ^Myosin  is  conveniently  isolated  from  muscle-plasma  by 
salting  with  ammonium  sulphate  to  the  extent  of  28  per  cent. 
Sodium  chloride  and  magnesium  sulphate  may  also  be  employed, 
but  it  is  then  necessary  to  add  the  salt  to  saturation. 

The  substance  is  a  globulin,  and,  curiously,  contains  a  consider- 
able amount  of  calcium.  It  is  soluble  in  dilute  saline  solutions,  and 
is  precipitated  from  these  solutions  by  salting,  as  just  indicated,  by 
passing  a  stream  of  carbon  dioxide  through  its  solutions,  by  diluting 
with  water,  and  on  dialysis.  It  is  characterized  bv  its  pronounced 
tendency  to  coagulate,  and,  unlike  myogen,  is  rendered  almost 
entirely  insoluble  on  precipitation  with  alcohol.  Like  this,  it  is  also 
readily  transformed  into  syntonin  or  alkaline  albuminate  on  treating 


376  THE  MUSCLE-TISSUE. 

with  acids  or  alkalies ;  and  here,  as  there,  a  precipitation  results 
only  if  very  dilute  acids  are  used.  In  an  excess  the  precipitate 
rapidly  dissolves.  On  heating  solutions  of  myosin  to  35°  C.  the 
substance  is  gradually  coagulated,  while  this  occurs  at  once  at  a  tem- 
perature of  50°  C. 

In  its  insoluble  form  myosin  is  termed  myosin-fihrin,  which,  like 
the  insoluble  myogen-fibrin,  belongs  to  the  class  of  the  coagulated 
albumins. 

Of  special  interest,  further,  is  the  fact  that  on  evaporating  a  few 
drops  of  a  solution  of  myosin  in  soda  solution  on  a  slide,  at  a  low 
temperature,  a  jelly-like  material  is  obtained,  which  on  polariscopic 
examination  is  seen  to  be  doubly  refracting.  In  this  respect  it 
behaves  exactly  as  the  anisotropic  material  which  is  found  in  the 
dark  bands  of  the  voluntary  muscle-fibres. 

The  amount  of  myosin  found  in  the  muscle-tissue  of  the  rabbit  is 
much  less  than  that  of  myogen,  and,  according  to  v.  Fiirth,  corre- 
sponds to  only  20  per  cent,  of  the  total  amount  of  solu])le  albumins. 

Significance  of  the  Common  Muscle -albumins. — Of  the  part 
which  the  common  muscle-albumins  take  in  the  function  of  the  cell 
little  is  known  that  is  definite.  From  the  researches  of  some  ob- 
servers, it  appears  that  the  nitrogenous  components  of  the  albumins, 
at  least,  do  not  furnish  the  energy  which  is  here  required.  Petten- 
kofer  and  Voit  have  thus  shown  that  an  increase  in  the  amount  of 
muscular  work  does  not  lead  to  an  increased  elimination  of  nitrogen 
or  to  an  increase  which  is  insignificant.  This  view  is  now  gener- 
ally held ;  but  it  must  be  admitted  that  evidence  is  not  lacking 
which  suggests  that  an  increased  albuminous  destruction  may  occur 
nevertheless  when  the  amount  of  work  is  increased.  It  has  been 
shown,  as  a  matter  of  fact,  that  the  total  elimination  of  sulphur, 
which  usually  follows  that  of  the  nitrogen  quite  closely,  is  increased 
by  muscular  exercise  and  diminished  thereafter.  But  while  we  may 
admit  that  the  nitrogenous  components  of  albumin  may  furnish  a 
certain  fraction  of  the  energy  which  is  recjuired  in  muscular  work, 
this  is,  after  all,  but  slight,  and  there  is  abundant  evidence  to  show 
that  by  far  the  greater  amount  of  energy  must  be  referable  to  the 
decomposition  of  non-nitrogenous  material. 

The  question,  of  course,  suggests  itself.  Do  the  soluble  albumins 
of  the  muscle-plasma  represent  the  contractile  element  of  the 
muscle-tissue?  but  to  this  question  no  answer  can  as  yet  be  given. 
We  might  imagine  that  in  some  manner  a  transformation  of  the 
soluble  albumins  into  the  fibrin  form  occurs,  and  vk-e  versa  ;  but  of  this 
we  have  no  evidence  in  the  living  tissue.  On  the  other  hand,  it  is 
supposed  that  rigor  mortis,  as  well  as  the  rigor  which  results  from 
exposure  of  muscle-tissue  to  a  temperature  of  47°  C,  is  owing 
to  such  a  change,  and  it  is  possible  that  in  either  event  both 
myosin  and  myogen  pass  over  into  tlie  coagulated  state.  Folin  in  a 
recent  j.aper,  however,  has  again  thrown  doubt  on  the  correctness 
of  the  coagulation  theory  in  the  explanation  of  rigor  mortis,  and  has 


THE  MUSCLE-ALBUMINS.  377 

oiFered  evidence  which  goes  to  show  that,  after  all,  the  process  may 
be  a  physical  one,  as  the  muscle-albumins,  according  to  his  experi- 
ments, take  no  part  in  the  process,  viz.,  there  is  no  coagulation  of 
the  muscle-albumins. 

Other  Albumins. — Besides  myosin  and  myogen,  which  latter  was 
formerly  termed  myos'inogen,  muscle-plasma  was  also  supposed  to 
contain  traces  of  serum-albumin,  myoglobulin,  and  myo-albvnnose. 
\.  Fiirth,  however,  has  shown  that  any  trace  of  serum-albumin  that 
may  be  found  is  referable  to  the  presence  of  small  amounts  of  lymph 
or  blood  that  have  not  been  removed  by  washing,  and  that  if  this  is 
done  with  special  care  no  serum-albumin  can  be  demonstrated. 
Halliburton's  myoglobulin,  moreover,  he  regards  as  identical  with 
myogen,  while  the  existence  of  a  myo-albumose  in  muscle-plasma 
has  been  disproved  by  more  recent  investigations.  That  substances 
belonging  to  the  albumoses  may  be  found  in  muscle-tissue  after  death, 
when  syntonin  also  is  found,  is,  of  course,  likely,  but  in  the  living 
tissue  their  presence  can  hardly  be  expected  under  normal  conditions. 

Myoproteid  is  a  substance  which  v.  Fiirth  obtained  from  the 
muscle-plasma  of  fish.  Of  its  chemical  nature,  however,  nothing 
further  is  known  than  the  fact  that  it  apparently  does  not  belong  to 
the  commonly  recognized  classes  of  albumins. 

Nucleoproteids  are  not  found  in  the  muscle-plasma,  but  can  be  iso- 
lated from  the  muscle-tissue  as  a  Avhole  or  from  the  insoluble  material 
which  remains  in  the  filter-press  after  separation  from  the  plasma. 
Their  amount  is  small,  and  in  accordance  with  the  slight  degree  to 
which  the  nuclei  enter  into  the  structural  composition  of  the  muscle- 
cell.  Larger  amounts  are  obtained  from  embryonic  muscle,  where 
cellular  reproduction  is,  of  course,  more  active.  From  the  tissue  of 
an  adult  dog  Pekelharing  obtained  about  0.37  per  cent.  These 
bodies  must  be  regarded  as  the  material  from  which  the  xanthin- 
bases  that  can  always  be  demonstrated  in  the  muscle-tissue  are  de- 
rived.    These  will  be  considered  in  detail  later. 

Phospho-camic  Acid. — Some  years  ago  Siegfrid  announced  that 
after  removing  the  phosphates  from  extracts  of  muscle-tissue, 
and  treating  with  ferric  chloride,  under  the  application  of  lieat,  a 
phosphorus-containing  iron  compound  is  obtained,  which  is  insolu- 
ble in  water,  but  easily  soluble  in  solutions  of  the  alkalies.  This 
substance  he  regards  as  the  iron  salt  of  an  organic  acid,  which  he 
terms  phosphor-carnic  acid  ;  the  salt  he  speaks  of  as  carniferrin. 
On  decomposition  with  barium  hydrate  he  then  obtained  the  barium 
salt  of  a  crystallizable  acid,  carnie  acid,  to  Avhich  he  gives  the 
formula  CjoHj^NgO^.  In  addition,  phosphoric  acid,  carbonic  acid, 
paralactic  acid,  succinic  acid,  and  a  substance  which  apparently 
belongs  to  the  carbohydrate  group,  are  found.  In  his  more  recent 
commimications  Siegfried  expresses  the  opinion  that  his  carnie  acid 
is  in  reality  pure  antipeptone.  This  question  is  still  under  debate, 
and  is  strongly  combated  by  Kutscher  and  others.  Kutscher, 
indeed,  has  shown  that  Kiihne's  antipeptone  is  in  reality  a  mixture 


378  THE  MUSCLE-TISSUE. 

of  different  substances,  and  he  has  demonstrated  tliat  it  can  be  sepa- 
rated into  two  fractions,  one  of  which  is  precipitated  by  phospho- 
tungstic  acid,  while  the  other  remains  in  solution.  In  the  first 
fraction  he  then  demonstrated  the  ])resence  of  hexon-bases,  while  in 
the  second  portion  mono-amido-acids  were  found.  Thus  far,  how- 
ever, only  a  fraction  of  the  antipeptone  has  been  resolved  into  sev- 
eral components,  and  we  must  admit  that  there  is  no  evidence 
to  show  that  the  remaining  portion  may  not  be  represented  by  a 
single  substance.  Whether  or  not  this  unresolved  portion  is  identical 
with  Siegfried's  carnic  acid,  however,  remains  to  be  seen.  If  so,  the 
remarkable  fact  would  be  demonstrated  that  a  peptone  may  occur  in 
crystalline  form. 

Panella  has  recently  pointed  out  that  phospho-carnic  acid  occurs  in 
much  larger  amounts  in  the  muscles  of  rabbits  than  in  those  of  dogs  ; 
that  the  amount  diminishes  after  death  in  proportion  to  the  appear- 
ance of  rigor  mortis  ;  and  that  it  increases  again  with  the  disappear- 
ance of  rigor  mortis  and  beginning  putrefaction. 

As  regards  the  significance  of  his  phospho-carnic  acid,  Siegfried 
expresses  the  opinion  that  it  may  serve  as  one  of  the  sources  of 
muscular  energy,  and  he  points  out  that  in  the  working  muscle  car- 
bonic acid  must  of  necessity  be  formed  on  hydrolysis  of  phosphor- 
carnic  acid  even  though  oxygen  be  absent.  In  this  manner  the 
observation  of  Hermann  would  be  explained,  viz.,  that  a  bloodless 
muscle  can  still  work  for  a  while  in  the  absence  of  oxygen  and  give 
off  carbon  dioxide.  The  lactic  acid  and  phosphoric  acid  which 
are  also  known  to  be  set  free  during  muscular  activity,  Siegfried 
likewise  refers,  in  })art  at  least,  to  a  hydrolytic  decomposition  of  his 
phosphor-earn ic  acid.  The  question,  however,  whether  the  carbo- 
hydrate and  ])hosphoric  acid  group  only  are  liberated,  he  leaves 
undecided. 

Of  the  chemical  nature  of  phosphor-earn ie  acid  little  is  known; 
but  it  is  manifestly  closely  related  to  the  nucleins,  and  is  accordingly 
termed  a  nucleon. 

Ferments. — Of  late,  Cohnheim  has  pointed  out  the  remarkable 
fact  that,  wliile  the  muscle-tissue  itself  does  not  contain  a  ferment 
wliich  is  capable  of  decomposing  the  large  amounts  (jf  glucose  M'hich 
are  daily  used  in  the  metabolism  of  the  muscles,  and  while  in  the 
pancreas  also  no  such  ferment  has  been  demonstrated,  extensive 
glucolysis  can  be  effected  by  a  mixture  of  fresh  pancreatic  extract 
and  the  muscle-plasma.  This  suggests  a  relation  between  two  pos- 
sible ferments  analogous  to  that  existing  between  trypsin  and  entero- 
kinase. 

Of  other  ferments  the  muscle-tissue  manifestly  contains  a  proteo- 
lytic ferment,  as  the  tissue  readily  undergoes  extensive  autolysis  after 
removal  from  the  body.  Rosell  claims  to  hav^e  isolated  a  trypsinoid 
ferment  of  this  order  with  the  uranyl-acetate  method.  In  addition 
to  these  ferments  we  find  evidence  of  a  ptyalin,  a  maltase,  and  a 
lactic-acid-producing  enzyme.      X  myosin-ferment,  which  might  be 


GLYCOGEN.  379 

responsible  for  the  development  of  rigor  mortis,  according  to  older 
concepts,  does  not  seem  to  exist  (v.  Fiirtli). 

Muscle-stroma. — Of  the  chemical  nature  of  the  so-called  muscle- 
stroma,  which  remains  after  the  extraction  of  the  soluble  albumins 
with  a  5  per  cent,  solution  of  ammonium  chloride,  we  know  only 
that  the  material  in  question  consists  of  an  albuminous  substance. 
It  is  apparently  a  native  albumin,  and,  like  the  soluble  muscle- 
albumins,  characterized  by  the  ease  with  which  it  is  transformed  into 
alkaline  albuminate  on  treating  with  dilute  solutions  of  alkalies. 

According  to  Danilewski  and  Holmgren,  the  structure  of  the 
muscle-fibre  is  in  no  ways  altered  by  dissolving  out  the  soluble 
albumins,  and  it  would  thus  appear  that  the  stroma  represents  the 
actual  contractile  substance  of  the  tissue.  Whether  or  not  this  is 
actually  the  case,  however,  is  as  yet  unknown. 

The  sarcolemma  apparently  consists  of  a  substance  Avhich  belongs 
to  the  albuminoids,  and  resembles  elastin  in  its  general  properties. 

THE   MUSCLE-PIGMENTS. 

As  I  have  already  indicated,  the  color  of  the  muscle-plasma  is  dif- 
ferent in  different  animals,  and  practically  coincides  with  the  color 
of  the  muscle-tissue  itself.  In  some  animals,  and  notably  the  mam- 
mals, this  is  dark  red,  while  the  muscles  of  others  are  almost  color- 
less. But  even  in  those  vertebrate  animals  in  which  no  color  is 
observed  in  the  skeletal  muscles  as  a  whole  the  heart-muscle  and 
the  diaphragm  always  aj)pear  dark  red.  This  difference  is  thought 
to  depend  upon  the  degree  of  activity  of  the  different  muscles,  but 
apparently  has  nothing  to  do  wdth  the  velocity  of  contraction  of 
which  a  muscle  is  capable. 

The  red-muscle  pigment  proper  is  now  known  to  be  identical 
with  the  hfemoglobin  of  the  blood,  and  probably  serves  the  same 
purpose,  as  a  carrier  of  oxygen,  in  the  internal  respiration  of  the 
tissue.  That  it  actually  occurs  within  the  cells  is  now  undoubted. 
Curiously  enough,  the  same  pigment  is  found  in  the  red  muscles  of 
certain  insects,  in  which  no  haemoglobin  otherwise  occurs. 

In  addition  to  haemoglobin  various  lipochromes  may  also  be 
encountered  in  muscle-tissue,  and  are  especially  abundant  in  certain 
fishes,  such  as  the  salmon  and  the  sea  trout.  Of  their  origin  and 
significance  nothing  is  known. 

GLYCOGEN. 

The  glycogen  which  is  found  in  muscle-tissue  does  not  occur  in 
the  body  of  the  cells  proper,  but  is  distributed  between  the  individual 
fibres  in  the  form  of  fine  threads,  which  are  apparently  connected  with 
the  connective-tissue  corpuscles. 

The  substance  is  formed  synthetically  in  the  muscle-tissue  through 
a   polymerization    of   the  anhydride  radicles  of  glucose,   which    is 


380  THE  MUSCLE-TISSUE. 

carried  to  the  tissue  either  directly  from  the  intestinal  tract,  or  which 
results  from  the  hepatic  glycogen  through  a  process  of  depoly- 
merization.  That  the  muscle-tissue  is  in  fact  capable  of  effecting  this 
synthesis  is  now  undoubted.  It  has  thus  been  shown  that  in  frogs 
a  deposition  of  glycogen  occurs  following  the  subcutaneous  injection 
of  a  solution  of  glucose,  even  after  removal  of  the  liver.  The 
amount  of  glycogen  which  is  deposited  in  the  muscle-tissue  probably 
represents  about  one-half  of  the  total  amount  that  is  found  in  the 
entire  body,  and  in  man  corresponds  to  150  grammes.  It  repre- 
sents the  most  important  source  of  energy  which  is  at  the  disposal 
of  the  tissue,  and  is  constantly  consumed,  even  when  the  muscle  is 
at  rest.  This  is  apparent  from  the  fact  that  after  section  of  the 
nerves  more  glycogen  is  found  in  a  given  muscle  than  in  the  cor- 
responding muscle  of  the  other  side,  while  ordinarily  this  is  the 
same.  While  at  work  the  consumption  of  glycogen  increases,  and 
after  a  comparatively  short  time  already  the  substance  has  entirely 
disappeared.  If  now  a  period  of  rest  follows,  glycogen  is  again 
stored  in  the  muscle,  and  so  on.  It  is  to  be  noted,  however,  that 
the  working  muscle  is  constantly  taking  up  sugar  from  the  blood, 
which  in  turn  is  derived  from  the  glycogen  of  the  liver,  and  that 
its  function  may  continue  even  though  a  deposition  of  glycogen,  as 
such,  does  not  occur.  This  shows  that  the  muscle  glycogen,  like 
that  of  the  liver,  is  in  reality  a  reserve  food,  and  is  here  deposited 
for  immediate  use.  In  starving  animals  it  gradually  disappears, 
but,  in  contradistinction  to  the  glycogen  of  the  liver,  its  supply  is 
not  exhausted  until  the  liver  itself  is  free  from  glycogen.  In  this 
connection  it  is  interesting  to  note  that  the  heart-muscle  still  has 
glycogen  at  its  disposal  under  conditions  when  the  skeletal  muscles 
are  already  free  from  glycogen,  viz.,  during  starvation  or  in  cases  in 
which  the  glycogen  is  rapidly  consumed  as  the  result  of  excessive 
work. 

The  chemical  changes  which  are  involved  in  the  transformation  of 
glycogen  into  glucose  are  probably  the  same  as  those  which  occut 
during  the  process  of  digestion.  JErythrodextrin  thus  first  results, 
and  is  then  transformed  into  achroodextrin,  and  this  into  maltose, 
which  in  turn  is  inverted  to  glucose.  This  is  then  supposedly  de- 
composed through  the  agency  of  a  specific  muscle-ferment,  activated 
by  a  pancreatic  kinase.  What  intermediate  products  are  formed  is 
not  known,  but  the  end-products  in  any  event  are  carbon  dioxide 
and  water.  The  amount  of  carbon  dioxide  that  is  eliminated  during 
a  period  of  exercise,  as  compared  with  one  of  rest,  may  serve  as  an 
index  of  the  amount  of  muscular  work  done.  I  have  already  pointed 
out  that  the  nitrogenous  constituents  of  the  muscle-tissue  cannot  be 
regarded  asasourceof  muscular  energy,  and  that  this  must  be  sought 
in  its  non-nitrogenous  componento.  This  fact  was  well  shown  during 
the  ascent  of  the  Faulhorn  mountain  by  Fickand  Wislicenus,  in  which 
it  was  calculated  that  the  total  amount  of  work  done  by  the  latter 
amounted  to  at  least  368,000  kilogrammeters.  The  amount  of  nitrogen 


GLYCOGEN.  381 

which  he  eliminated  during  the  ascent  and  the  six  hours  following 
corresponded  to  37  grammes  of  albumin.  Translated  into  calories,  this 
would  represent  about  106,000  kilogrammeters  of  work.  Deducting 
this  from  368,000,  there  would  remain  262,000  kilogrammeters, 
which  could  not  be  accounted  for  by  a  decomposition  of  nitrogenous 
material,  and  which  must  hence  be  referable  to  the  destruction  in  the 
muscles  of  other  bodies  which  are  free  from  nitrogen.  Of  these,  the 
glycogen  which  is  referable  to  ingested  carbohydrates  is  no  doubt  the 
most  important.  But  while  normally  the  muscle  glycogen  is  prob- 
ably derived  from  this  source  exclusively^  there  is  evidence  to  show 
that  it  may  be  .formed  from  the  albumins  as  well.  If  animals  are 
allowed  to  starve  until  the  entire  reserve  of  glycogen  has  been  con- 
sumed, and  they  are  then  fed  on  albumins  exclusively,  it  will  be 
observed  that  a  gradual  deposition  of  glycogen  occurs  nevertheless, 
which  can  be  referable  only  to  the  ingested  albumins.  In  the  severer 
forms  of  diabetes,  moreover,  as  will  be  shown  later,  sugar  appears 
in  the  urine  although  all  carbohydrates  are  excluded  from  the 
diet.  If  then  in  such  cases  the  nitrogenous  metabolism  is  diminished 
as  much  as  possible  by  reducing  the  albumins  of  the  food  to  a  mini- 
mum, and  a  day  of  fasting  is  further  introduced  into  the  regime,  or 
when  diarrhoea  occurs,  the  amount  of  sugar  that  appears  in  the  urine 
is  notably  reduced.  If,  on  the  other  hand,  the  amount  of  albumin 
in  the  food  is  increased,  an  increased  elimination  of  sugar  occurs  ; 
the  parallelism  between  the  elimination  of  nitrogen  and  sugar  may 
then  be  quite  remarkable.  At  times  the  ratio  of  nitrogen  to  sugar 
has  been  found  to  be  1  :  3  to  1:4.  Similar  conditions  prevail  in 
cases  of  experimental  diabetes  folloAving  extirpation  of  the  pancreas 
or  poisoning  by  phloridzin. 

The  manner  in  which  the  glycogen,  viz.,  the  glucose,  is  formed 
under  such  conditions  has  long  been  a  matter  of  speculation.  That 
the  carbohydrate  group  of  the  albumins  does  not  play  an  important 
role  in  this  connection  is  apparent  at  once,  as  the  amount  is  far  too 
small  to  account  for  the  large  amount  of  glucose,  which  may  still  be 
eliminated  in  the  urine  in  severe  diabetes  at  a  time  when  no  carbo- 
hydrates are  administered  in  the  food.  Benedix,  moreover,  has 
shown  that  in  dogs  which  are  fed  on  casein  (in  which  a  carbohydrate 
group  does  not  exist)  glycogen  is  nevertheless  stored  in  the  liver  in 
notable  amounts.  Under  such  circumstances  the  leucin  complex 
may  be  the  source  of  the  glucose,  with  the  intermediate  formation 
of  oxy-capronic  acid.  This  possibility  has  been  suggested  by  Miiller, 
Seeman,  and  Cohn.  Opposed  to  this  view  is  the  fact  that  the  leucin 
of  the  tissues  contains  a  divided  chain  of  carbon  atoms,  being  the 
isobutyl-amido-capronic  acid.  But,  on  the  other  hand,  Miiller  has 
pointed  out  the  readiness  with  which  glucose,  with  its  uninterrupted 
chain,  will  pass  over  into  tetraoxy-capronic  acid  on  standing  in  con- 
tact with  calcium  hydrate.  This  latter  has  a  divided  chain,  and  we 
can  thus  imagine  ]:)erfectly  well  that  the  reverse  also  can  happen. 

Of  other  possibilities  which  suggest  themselves  there  are  two,  both 


382  THE  MUSCLE-TISSUE. 

of  which  may  be  operative  at  the  same  time.  On  the  one  hand,  we 
may  imagine  that  CHOH  groups  are  split  off  directly  from  the 
albuminous  molecule  and  are  then  condensed  to  glucose;  while  on 
the  other,  it  is  conceivable  that  the  albuminous  molecule  may  con- 
tain atomic  groups  which  under  certain  conditions  may  be  changed 
to  glucose  directly  without  the  occurrence  of  an  actual  synthesis. 
Opposed  to  this  last  possibility  is  our  present  conception  of  the  pre- 
existence  of  the  various  radicles  which  are  obtained  on  hydrolysis. 

Whetlier  or  not  the  fats  also  can  give  rise  to  the  formation  of 
glycogen  has  not  been  established  beyond  doubt.  It  seems,  however^ 
that  this  does  not  occur.  Liittje  has  thus  shown  in  4  severe  case  of 
diabetes  that  while  the  patient  eliminated  60  grammes  of  nitrogen 
and  112  grammes  of  sugar  while  receiving  400  grammes  of  nutfose, 
the  nitrogen  fell  to  9.9  grammes  and  the  sugar  disappeared  alto- 
gether, when  three  day  later  the  albuminous  metabolism  was  dimin- 
ished as  much  as  possible  by  giving  but  little  albumin,  but  furnisii- 
ing  much  fat.  To  a  certain  extent  the  fats  can  supply  the  energy, 
however,  which  is  necessary  for  the  functioning  of  muscle-tissue  when 
a  sufficient  supply  of  glycogen  is  not  available. 

While  I  have  stated  above  that  as  a  result  of  muscular  activity 
the  glucose  wiiich  is  derived  from  the  muscle  glycogen  is  decora- 
posed  into  carbon  dioxide  and  water,  there  is  evidence  to  show  that 
this  decomposition  does  not  occur  in  the  sense  of  a  direct  oxidation. 
It  is  hence  assumed  that  a  primary  splitting  up  of  the  glucose 
molecule  occurs,  but  of  the  products  which  are  formed  nothing 
definite  is  known.  On  the  one  hand,  we  may  supjiose  that  lactic 
acid  thus  results,  but  we  may  also  imagine  that  alcohol  is  produced, 
and  is  then  oxidized  to  carbon  dioxide  and  water.  As  a  matter  of 
fact,  traces  of  alcohol  are  always  found  when  perfectly  fresh  organs 
are  distilled  with  water  immediately  after  their  removal  from  the 
body. 

Of  the  role  which,  the  pancreas  plays  in  the  sugar  metabolism  of 
muscle-tissue  mention  has  already  been  made.  In  the  formation  of 
the  glycogen  and  its  inversion  to  glucose  ferments  are  also  no  doubt 
active. 

Isolation. — If  it  is  desired  to  isolate  the  glycogen  from  muscle- 
tissue,  it  is  necessary  to  place  the  material  in  boiling  water  imme- 
diately after  the  death  of  the  animal,  so  as  to  prevent  its  trans- 
formation into  glucose  and  the  resulting  products  of  decomposition. 
Otherwise  this  will  occur,  as  the  death  of  the  individual  cells  does 
not  coincide  in  point  of  time  with  the  death  of  the  animal  as  a 
whole,  and  there  is  danger,  moreover,  that  the  inverting  ferments 
of  the  tissue  remain  active.  That  this  actually  occurs  can  be 
readily  demonstrated  by  treating  one  portion  of  the  muscle-tissue  as 
described,  while  a  second  portion  is  allowed  to  remain  exposed  to 
the  air  for  a  few  hours.  Both  portions  are  then  examined  for 
glycogen,  when  it  will  be  seen  that  only  the  first  gives  a  positive 
reaction.     (As  regards  the  details  of  the  method,  sec  page  435-) 


LACTIC  ACID.  383 

Quantitative  Estimation  (according  to  Pfliiger). — 100  grammes  of 
finely  liashed,  perfectly  fresh  muscle-tissue  and  100  c.c.  of  a  60 
per  cent,  solution  of  potassium  hydrate  (Merck  la)  are  shaken  in  a 
200  c.c.  flask,  so  that  the  meat  is  evenly  distributed.  The  mixture 
is  heated  for  two  hours  on  a  boiling  water-bath,  then  transferred 
to  a  400  c.c.  flask,  diluted  to  the  400  c.c.  mark,  and  filtered  through 
glass  wool.     The  filtrate  should  be  clear  or  but  faintly  opalescent. 

A  carefully  measured  portion  of  100  c.c.  of  the  filtrate  is  treated 
with  an  equal  quantity  of  96  per  cent,  alcohol.  After  standing  for 
twenty-four  hours  the  precipitate  is  collected  on  a  15  c.c.  filter 
(Swedish  Munktell's),  first  washed  with  a  mixture  of  1  volume  of 
15  per  cent,  potassium  hydrate  solution  and  2  volumes  of  96  per 
cent,  alcohol  and  then  with  the  alcohol  alone.  It  is  now  dissolved 
in  water,  the  solution  accurately  neutralized  with  hydrochloric 
acid,  transferred  to  a  500  c.c.  flask,  treated  with  25  c.c.  of  hydro- 
chloric acid  (specific  gravity  1.19)  and  almost  diluted  to  the  mark. 
Should  the  amount  of  glycogen  be  very  small,  as  much  as  300  c.c. 
of  the  original  filtrate  must  be  used.  The  flask  is  closed  and 
heated  for  three  hours  on  a  boiling  water-bath.  Water  is  then 
added  to  the  500  c.c.  mark  and  the  sugar  estimated  according  to 
the  method  which  Pfliiger  describes.  For  a  consideration  of  this 
method  the  reader  is  referred  to  the  original  (FJlUger's  Arch.,  1902, 
vol.  93,  p.  163). 

GLUCOSE. 

That  traces  of  glucose  and  maltose  may  be  found  in  fresh  muscle- 
tissue  is,  of  course,  not  surprising  in  view  of  the  above  considerations. 
To  demonstrate  their  presence,  the  fresh  material  is  finely  hashed, 
placed  in  boiling  water,  and  boiled  for  a  few  minutes.  On  cooling, 
the  mixture  is  filtered,  the  filtrate  concentrated  to  a  small  volume 
and  examined  in  the  usual  manner.  Larger  quantities  may  be 
obtained  if  the  finely  minced  tissue  is  placed  in  chloroform- water 
and  autodigestion  is  allowed  to  proceed  for  several  weeks. 

LACTIC  ACID. 

The  reaction  of  living  muscle-tissue  while  at  rest  is  neutral  or 
slightly  alkaline.  After  death,  however,  it  becomes  acid,  and  it 
can  then  be  demonstrated  that  the  acidity  is  in  part,  at  least,  refer- 
able to  paralactic  acid.  Through  the  action  of  the  latter  upon 
dipotassium  phosphate  monopotassium  phosphate  then  results,  and 
a  second  factor  thus  appears,  to  which  the  acid  reaction  is  due. 

Formerly  it  was  supposed  that  rigor  mortis  was  the  result  of  the 
formation  of  lactic  acid,  but  we  now  know  that  this  is  not  the  case, 
and  that  the  coagulation  of  the  muscle-tissue  precedes  the  appearance 
of  the  acid  reaction.  To  use  the  words  of  Salkowski,  the  muscle  does 
not  form  lactic  acid  because  it  dies,  but  because  it  lives,  and  only  as 
Ions'  as  it  lives.    With  the  occurrence  of  its  death  the  formation  ceases. 


384  THE  MUSCLE-TISSUE. 

This  is,  therefore,  a  vital  or  nltravital  process,  and  there  :s  abundant 
evidence  to  show  that  this  view,  which  is  now  quite  generally  acee])ted, 
is  correct.  Under  ordinary  conditions  it  is  difficult  to  show  that 
acid  material  is  produced  while  the  muscle  is  at  work,  as  it  is  then 
removed  by  the  circulation  as  rapidly  as  formed ;  but  if  this  is 
prevented,  the  fact  can  readily  be  demonstrated.  To  this  end,  one 
sciatic  nerve  of  a  rabbit  is  divided  and  the  animal  poisoned  with 
strychnin.  If  then  the  muscles  of  both  legs  are  removed  during  the 
final  convulsions  of  the  animal,  it  will  be  noted  that  the  reaction  of 
those  groups  which  had  remained  in  connection  with  their  nerve- 
supply  is  distinctly  acid,  w^hile  the  others,  the  nerve  of  which  was 
severed,  show  a  neutral  reaction.  Tliat  the  acid  reaction  in  such 
cases  is,  in  part  at  least,  actually  due  to  lactic  acid,  can  be  shown  by 
extracting  the  rested  and  the  tetanized  groups  with  water  and  then 
with  alcohol.  On  evaporating  the  resulting  extracts  and  weighing 
the  corresponding  residue  it  will  be  noted  that  the  weight  of  the 
alcoholic  fraction  is  greater  in  the  case  of  the  worked  muscle  than 
of  those  that  have  rested,  while  the  reverse  holds  good  for  the 
aqueous  portions. 

Lactic  acid  is,  however,  produced  by  the  muscle  not  only  when 
at  work,  but  also  while  resting.  This  has  been  shown  by  Zillessen 
and  V.  Frey.  These  observers  found  that  on  transfusing  the  muscles 
of  the  hindquarters  of  a  dog,  during  three  hours,  an  increase  in  the 
amount  of  lactic  acid  resulted,  which,  calculated  for  the  entire  amount 
of  blood,  corresponded  to  as  much  as  1.48  grammes  of  zinc  lactate. 

The  amount  of  lactic  acid  that  may  be  isolated  from  dead  muscles 
wdiile  still  rigid  varies  between  0.1  and  1.0  per  cent.,  and  it  is  note- 
worthy that  for  definite  groups  of  muscles  this  amount  is  constant, 
no  matter  whether  the  formation  of  the  acid  is  allowed  to  proceed 
rapidly  or  slowly.  This,  however,  holds  good  only  for  correspond- 
ing muscles,  and  is  different  in  different  groups.  In  rabbits  larger 
amounts  can  thus  always  be  obtained  from  the  muscles  of  the  trunk 
than  from  those  of  the  extremities. 

To  the  general  rule  that  the  acidity  of  corresponding  muscles  is 
always  the  same,  there  is  one  exception,  viz.,  the  heart,  which  is  the 
only  muscle  of  the  body,  moreover,  that  normally  presents  an  acid 
reaction.  Larger  amounts  of  lactic  acid  are  here  always  found  in 
the  left  than  in  the  right  side. 

As  regards  the  origin  of  lactic  acid  in  muscle-tissue,  it  w^as  long 
thought  that  the  glycogen  probably  represented  its  principal  source. 
There  area  number  of  facts  indeed  which  favor  such  an  assumption. 
I  have  pointed  out  already  that  after  death  tlie  glycogen  gradu- 
ally disappears,  and  we  have  just  seen  that  lactic  acid  is  then  found. 
Glycogen  is  similarly  decomposed  during  muscular  activity  in  the 
living  animal,  where  lactic  acid  is  also  constantly  produced,  and,  as 
I  have  shown,  the  same  also  occurs  in  tlie  muscle  while  at  rest. 
Then  again  there  is  evidence  to  show  that  the  decomposition  of 
glucose  in  the  muscle-tissue  does  not  occur  in  the  sense  of  a  direct 


LACTIC  ACID.  385 

oxidation,  but  tliat  a  primary  division  of  the  molecule  occurs,  and 
that  lactic  acid  may  be  one  of  the  resulting  products.  But,  on  the 
other  hand,  observations  exist  which  go  to  show  that  the  amount  of 
lactic  acid  that  is  produced  during  rigor  mortis  bears  no  relation  to 
the  amount  of  glycogen  which  was  present  at  the  time,  and  it  has 
further  been  noted  that  lactic  acid  is  still  formed  in  muscles  from 
which  all  glycogen  has  previously  been  removed  by  starvation.  The 
conclusion  hence  suggests  itself  that  while  a  certain  amount  of 
lactic  acid  may  be  derived  from  glycogen,  this  does  not  represent  its 
only  source,  and  we  must  admit  that  to  some  extent  the  albumins 
of  muscle-tissue  also  contribute  toward  its  formation.  There  is  a 
tendency  among  physiological  chemists  at  the  present  time  to  regard 
this  source  indeed  as  the  most  important.  This  view  is  largely  based 
upon  observations,  which  go  to  show  that  increased  amounts  of  lactic 
acid  appear  in  the  urine  whenever  the  formation  of  urea  is  impaired 
in  the  liver,  or  when  this  organ  is  entirely  excluded  from  the  gen- 
eral circulation.  Similar  results  also  have  been  obtained  in  birds, 
in  wdiich  uric  acid  represents  the  most  important  end-product  of  the 
normal  nitrogenous  metabolism.  In  geese  it  could  ha  demonstrated 
that  after  removal  of  the  liver  the  elimination  of  lactic  acid  was  in 
no  ways  influenced  by  an  increased  or  diminished  ingestion  of  carbo- 
hydrates, while  the  administration  of  larger  amounts  of  albumin 
invariably  results  in  a  corresponding  increase  of  the  lactic  acid. 
When  from  any  reason,  moreover,  albuminous  decomposition  is  in- 
creased, while  the  oxidation-processes  of  the  body  are  at  the  same 
time  diminished,  increased  amounts  of  lactic  acid  are  found  in  both 
the  blood  and  the  urine. 

We  have  seen  that  Siegfried's  phosphor-carnic  acid  gives  rise 
to  the  formation  of  lactic  acid  on  liydrolytic  decomposition,  and  it 
is  thus  possible  that  this  substance  may  be  its  immediate  antecedent. 
Further  researches,  however,  are  necessary  before  the  formation  of 
lactic  acid  from  the  muscle-albumins  can  be  satisfactorily  explained. 
Whether  or  not  enzymatic  influences  are  here  at  ^vork  we  do  not 
know\  Salkowski  denies  this  possibility  on  the  basis  that  lactic  acid 
is  not  found  among  the  products  of  autodigestion  when  perfectly 
fresh  muscle-tissue  is  allowed  to  stand  in  contact  with  chloroform- 
water,  as  the  chloroform,  according  to  this  observer,  does  not  pre- 
vent the  action  of  enzymes.  If  this  property  holds  for  all  ferments, 
the  conclusion  would  also  follow  that  the  formation  of  lactic  acid 
can  neither  be  referable  to  the  action  of  living  protoplasm,  as  the 
chloroform  represents  a  strong  protoplasmatic  poison.  We  have 
seen,  as  a  matter  of  fact,  that  muscle-plasma  also  becomes  acid  after 
the  occurrence  of  coagulation,  and  protoplasmatic  activity  here 
manifestly  does  not  enter  into  consideration.  We  are  hence  forced 
to  the  conclusion  that  the  formation  of  lactic  acid  is  either  referable 
to  the  action  of  a  ferment  which  is  destroyed  by  chloroform,  or  that 
it  results  from  a  spontaneous  decomposition  of  certain  substances 
which  are  especially  unstable. 


386  THE  MUSCLE-TISSUE. 

Besides  paralaetic  acid,  traces  of  common  lactic  acid  also  are  said 
to  occur  in  muscle-tissne.  To  isolate  the  bodies  in  question,  the  fol- 
lowino-  j)r()cc(hu'c  may  l)e  employed. 

Isolation  and  Quantitative  Estimation. — A  carefully  weighed 
amount  of  muscle-tissue  is  finely  hashed,  repeatedly  extracted  with 
cold  water,  and  the  mixture  passed  through  a  muslin  filter.  The 
resulting  fluid  is  feebly  acidified  with  sulphuric  acid  and  boiled, 
so  as  to  remove  the  coas^ulable  albimiins.  Baryta-Avater  is  now 
added  so  long  as  a  precipitate  is  formed  ;  this  is  filtered  oft'.  The 
filtrate  is  freed  from  the  barium  that  was  added  in  excess  by  pass- 
ing carbon  dioxide  into  the  solution,  when  it  is  boiled,  filtered,  and 
concentrated  to  a  thin  syrup.  Care  should  be  taken,  however,  that 
the  temperature  does  not  exceed  70°  C.  toward  the  end.  The  re- 
sulting material  is  treated  with  ten  times  its  volume  of  al)Solute 
alcohol,  set  aside  for  a  while,  and  filtered.  The  alcoholic  solution  is 
evaporated  on  a  water-bath,  and  the  remaining  thick  syrup  treated 
with  about  an  equal  amount  of  a  moderately  dilute  solution  of  phos- 
phoric acid.  This  liberates  the  lactic  acid  from  its  salts,  while  the 
chlorides  and  sulphates  remain  unafiected.  The  lactic  acid  is  then 
extracted  with  ether.  The  ether  is  distilled  oft",  the  residue  boiled 
with  water  and  an  excess  of  carbonate  of  zinc,  and  filtered  while  still 
hot.  The  filtrate  is  concentrated  to  a  small  volume,  when  on  stand- 
ing, and  especially  after  the  addition  of  a  small  amount  of  alcohol,, 
the  zinc  lactate  crystallizes  out.  To  separate  the  paralactate  from 
the  common  lactate,  which,  as  I  have  said,  is  also  found  in  traces  in 
the  muscle-tissue,  the  crystals  are  placed  in  absolute  alcohol,  which 
dissolves  the  paralactate  (solubility  1  :  1100),  while  the  common  form, 
is  insoluble.     They  are  then  finally  dried  and  weighed. 

To  obtain  the  lactic  acid  as  such  the  lactates  are  decomposed  with 
hydrogen  sulphide.  The  resulting  zinc  sulphide  is  filtered  off", 
washecl  with  water,  when  filtrate  and  washings  are  evaporated  at 
70°  C.  to  a  small  volume. 

Both  acids  are  amorphous,  and  are  obtained  in  the  form  of  a  thick 
syrup,  which  is  soluble  in  water,  alcohol,  and  ether.  Of  their  salts^ 
the  zinc  salts  are  especially  characteristic  and  serve  to  distinguish 
the  two  forms  from  each  other.  As  I  have  indicated,  the  common 
lactate  is  insoluble  in  absolute  alcohol.  It  crystallizes  with  three 
molecules  of  water,  which  escape  at  a  temperature  of  105°  C,  so 
that  the  loss  of  weight  will  then  correspond  to  18.18  per  cent.  The 
paralactate,  on  the  other  hand,  is  soluble  in  absolute  alcohol,  though 
with  difticulty,  and  crystallizes  out  with  only  two  molecules  of  water, 
which  likewise  escapes  at  105°  C.  In  this  case  the  loss  of  weight 
amounts  to  12  per  cent.  Both  the  free  acid  and  the  paralactate  are 
Isevorotatory,  while  the  common  form  and  its  salts  are  optically 
inactive. 


INOSIT.  387 


INOSIT. 


Of  the  origin  of  iiiosit,  which  is  apparently  a  constant  constituent 
of  muscle-tissue,  but  which  is  found  also  in  other  organs  of  the 
body,  and  appears  in  the  urine  when  polyuria  is  either  artificially 
produced  or  results  from  some  morbid  process,  nothing  is  known. 
It  is  not  peculiar  to  the  animal  world,  however,  but  occurs  widely 
distributed  in  the  vegetable  kingdom  also,  and  is  identical  with  the 
so-called  phaseo-mannite,  which  is  especially  abundant  in  certain 
beans.     In  muscle-tissue  it  is  found  only  in  traces. 

The  substance  is  not  a  carbohydrate,  as  was  once  supposed,  but 
belongs  to  the  aromatic  series,  and  is  commonly  regarded  as  hexa- 
hydroxybenzol : 

CH.OH 

OH.H(y^^CH.OH 

I  I 

OH.HC.   ^CH.OH 

CH.OH 

In  pure  form  it  crystallizes  in  colorless,  monoclinic  prisms,  which 
are  often  grouped  in  rosettes.  It  melts  at  217°  C.  It  is  soluble 
in  water  and  dilute  alcohol,  but  is  insoluble  in  absolute  alcohol  and 
ether.  The  substance  does  not  reduce  the  metallic  oxides  in  alka- 
line solution,  and  is  optically  inactive.  It  is  not  fermentable  with 
common  yeast,  but  is  decomposed  by  the  Bacterium  lactis  with  the 
formation  of  lactic  acid,  and  subsequently  yields  butyric  acid. 

Tests. — Scherer's  Test. — If  a  few  crystals  of  inosit  are  evaporated 
on  platinum  foil  with  a  little  nitric  acid,  and  the  residue  is  treated 
with  ammonia  and  a  drop  of  dilute  solution  of  calcium  chloride,  a 
rose-red  spot  remains  on  further  evaporation.  The  reaction  is  due 
to  the  formation  of  rhodizonic  acid. 

'  Gallois'  Test. — On  evaporating  a  small  amount  of  a  solution  of 
inosit  and  adding  a  few  drops  of  a  dilute  solution  of  mercuric 
nitrate  before  the  residue  has  become  dry,  a  yellow  spot  develops 
on  the  further  application  of  heat,  which  ultimately  turns  red.  (3n 
cooling,  the  red  color  disappears,  but  reappears  on  heating. 

Isolation. — To  demonstrate  the  presence  of  inosit  in  muscle- 
tissue,  this  is  finely  hashed,  and  extracted  with  hot  water,  when  the 
albumins  are  removed  by  boiling.  The  filtrate  is  precipitated  with 
barium  hydrate,  so  as  to  remove  the  phosphates  that  are  present. 
After  filtering  the  liquid  is  then  concentrated,  until  most  of  the 
kreatin  has  separated  out.  This  is  removed  by  filtration ;  the  fil- 
trate is  boiled  with  four  times  its  volume  of  alcohol ;  the  solution 
is  allowed  to  cool,  freed  from  the  mineral  constituents  that  have 
separated  out,  and  shaken  with  ether.  The  inosit  then  separates 
out  in  the  form  of  fine  platelets,  which  can  be  further  purified  by 
dissolution  in  alcohol  and  reprecipitation  with  ether. 

The  scyllite  which  is  found  in  cartilaginous  fish,  where  inosit 
is  absent,  is  closely  related  to  the  latter,  and,  like  this,  gives  the 
reaction  of  Gallois. 


388  THE  3IUSCLE-TISSUE. 


THE   NITROGENOUS   EXTRACTIVES. 

The  nitrogenous  extractives  of  muscle-tissue  comprise  the  com- 
mon derivatives  of  the  nuclear  nucleins,  viz.,  xanthin,  hypoxanthin, 
guanin,  and  carnin ;  further,  traces  of  taurin,  glycocoll,  urea,  and 
uric  acid ;  more  abundantly  kreatin  and  kreatinin,  and  finally  a 
basic  substance  which  has  been  isolated  from  beef  extract,  and  Mhich 
Gulewitsch  has  termed  carnosin. 

Kreatin  and  Kreatinin. — Kreatin  is  a  constant  constituent  of 
the  muscle-tissue  of  the  vertebrate  animals,  while  in  the  invertebrates 
it  has  not  as  yet  been  found.  Its  amount  is  often  quite  considera- 
ble, and  it  has  been  calculated  that  in  adult  man  as  much  as  90 
grammes  could  be  extracted  from  the  muscles  of  the  entire  body. 
Its  anhydride,  kreatinin,  on  the  other  hand,  is  usually  found  only 
in  traces,  but  may  occur  in  larger  amounts,  and  notably  in  certain 
fishes. 

Of  the  origin  of  kreatin  little  is  known,  and  it  is  noteworthy 
that  the  substance  has  thus  far  not  been  obtained  from  the  animal 
tissues  directly  by  artificial  means.  It  has  been  found  in  the  brain, 
in  the  thyroid  gland,  in  the  blood,  in  transudates,  in  the  amniotic 
fluid,  and  is  also  a  constant  constituent  of  the  urine.  In  the  vege- 
table world  it  does  not  occur. 

From  the  observation  of  St.  Johnson  that  the  urinary  kreatinin 
is  not  identical  with  the  substance,  which  can  be  isolated  from 
muscle-tissue,  it  has  been  concluded  that  the  former  may  not  be 
derived  from  the  muscles  at  all,  but  may  possibly  be  referable  to  the 
kreatin,  which  is  found  in  other  organs  of  the  body  and  notably  in 
the  thyroid  gland.  Its  identity  with  these  kreatinins,  however,  has 
not  yet  been  established,  nor  is  there  reason  to  suppose  that  these 
forms  differ  from  the  common  kreatinin  of  the  muscles.  However 
this  may  be,  the  kreatius,  viz.,  kreatinins,  are  essentially  specific- 
decomposition-products  of  muscle-tissue,  and  are  unquestionably 
derived  from  the  common  muscle-albumins.  This  is  suggested  by 
the  observation  that  larger  amounts  of  kreatin  and  kreatinin  can  be 
isolated  from  muscles  that  have  previously  been  worked,  than  from 
muscles  that  have  been  at  rest.  I  have  shown,  it  is  true,  that  the 
nitrogenous  components  of  the  muscle-tissue  enter  into  considera- 
tion only  in  a  secondary  manner,  as  a  source  of  muscular  energy, 
but  this  does  not  preclude  the  possibility,  that  during  w^ork  the 
metabolism  of  the  muscle-albumins  is  increased.  That  such  an 
increase  w'ill  of  necessity  become  more  apparent  during  starvation  is, 
of  course,  self-evident,  and  we  find,  as  a  matter  of  fact,  that  under 
such  conditions  a  corresponding  increase  in  the  formation  of  kreatin 
occurs. 

While  our  knowledge  of  the  manner  in  which  kreatin  and 
kreatinin  are  produced  in  the  body  is  as  yet  practically  nil,  we  know, 
on  the  other  hand,  that,  w^hen  once  formed,  it  is  further  decomposed, 
and  contributes  toward  the  formation  of  urea.     Artificially  this  can 


THE  NITROGENOUS  EXTRACTIVES.  389 

readily  be  accomplished  by  boiling  kreatin  with  baryta-water,  when 
it  is  decomposed  with  the  formation  of  urea  and  methyl-glycocoll. 
At  the  same  time,  however,  methyl-hydantoin  and  ammonia  result. 
Whether  or  not  the  decomposition  of  kreatin,  which  is  now  known 
to  occur  in  the  muscle-tissue  itself,  takes  place  in  the  same  manner, 
is  not  known.  But  if  so,  we  could  readily  understand  why  traces 
of  glycocoll  are  also  so  constantly  met  with.  Both  substances,  how- 
ever, are  manifestly  removed  as  rapidly  as  possible,  as  neither  urea 
nor  glycocoll  is  ever  found  in  the  tissue  itself  beyond  traces.^ 

In  this  connection  it  is  interesting  to  note  that  GuareschI  and 
Mosso  have  succeeded  in  extracting  methyl-hydantoin  also  from  the 
muscles  of  the  calf.  That  methyl-hydantoin  belongs  to  the  class  of 
ureids  has  already  been  pointed  out,  and  we  may  therefore  assume 
that  the  substance  is  further  decomposed  with  the  formation  of 
urea,  if  further  researches  should  show  that  the  decomposition  of 
kreatin  actually  takes  place  in  the  living  tissue  also,  as  outlined 
above.  The  small  amount  of  ammonia,  which  at  the  same  time 
results,  possibly  combines  with  lactic  acid,  and  is  then  further  trans- 
formed into  urea  in  the  liver. 

While  the  kreatin,  which  is  thus  produced  during  the  nitrogenous 
metabolism  of  the  muscle-tissue,  is  largely  transformed  into  urea,  it 
is  noteworthy  that  the  substance,  when  ingested  by  the  mouth, 
reappears  in  the  urine  in  practically  the  same  amount.  This  ob- 
servation is  explained  by  the  assumption  that  the  kreatin  in  this 
case  does  not  pass  through  the  muscles,  and  thus  escapes  decom- 
position, and  there  can  be  no  doubt  that  a  certain  fraction  of  the 
kreatin  which  is  eliminated  in  the  urine  is  referable  to  this  source. 

Properties. — Kreatin  crystallizes  in  rhombic  prisms,  Avith  one 
molecule  of  water,  which  escapes  at  100°  C.  It  is  readily  soluble 
in  warm  water,  less  so  in  cold  water,  and  is  insoluble  in  alcohol  and 
ether. 

As  has  been  indicated  before,  it  can  be  formed  synthetically  from 
cyanamide  and  methyl-glycocoll,  according  to  the  equation  : 

CN.NH2   +  CH2.NH(CH3).COOH    =   NH  :   C< 

Cyanamide.  Methyl-glycocoll.  ^NfCHaj.CHj.COOH 

Kreatin. 

On  boiling  its  acidified  aqueous  solutions,  the  substance  loses 
water  and  is  transformed  into  kreatinin,  from  which  the  kreatin  is 
again  obtained  by  treating  with  dilute  alkaline  solutions.  The 
transformation  of  kreatinin  into  kreatin  can  indeed  take  place  in  the 
aqueous  solution  directly,  and  may  be  hastened  by  the  application 
of  heat.  The  relation  between  the  two  substances  can  thus  be 
illustrated  by  the  equation : 

^  This  statement  requires  modification,  as  quite  considerable  amounts  of  urea 
can  be  demonstrated  in  the  muscles  of  certain  fishes,  such  as  the  shark  and  the 
sturgeon. 


390  THE  MUSCLE-TISSUE. 


NH  :  C/       *  =  NH  :   c/       '^^----^        +  H,0 

\N(CH3).CH2.C00H  \N(CH3).CH2.C0 

Kreatin.  Kreatiniu. 

The  same  relation  thus  exist^^  betAveen  kreatin  and  kreatiniu  as 
between  glucocyaniin  and  glucocyamidin,  and,  as  a  matter  of  fact, 
both  are  methyl-substitution-products  of  the  two  latter,  and  are 
accordingly  also  termed  methyl-glucocyamin  and  methyl-glucocy- 
amidin. 

NH  :   Ck;  NH  :    C< 

\nh.ch,.cooh  ^NCCHO.CHj.COOH 

Glucocyamin.  Kreatin. 

/NH— CO  /NH^_ 

NH  :   C<  1  NH  :    C  ^^^^ 

\NH.   CH;,.  \N(CH3).CH2-C0 

Glucocyamidin.  Kreatinin. 

Kreatinin  crystallizes  in  prisms,  without  water  of  crystallization, 
and  is  soluble  in  water  and  alcohol  (see  also  pages  1 08  and  263). 

Isolation  of  Kreatin. — To  isolate  kreatin  from  muscle-tissue,  this 
is  finely  hashed  and  repeatedly  extracted  with  an  equal  weight  of 
water  at  a  temperature  of  from  55°  to  60°  C.  The  extracts  are 
boiled  so  as  to  remove  coagulable  albumins.  The  filtrate  is  pre- 
cipitated with  subacetate  of  lead,  care  being  taken  to  avoid  an 
excess.  The  resulting  filtrate  is  freed  from  lead  by  hydrogen  sul- 
phide, and  is  concentrated  to  a  small  volume  at  as  low  a  temperature 
as  possible.  On  standing,  the  kreatin  crystallizes  out,  and  can  then 
be  purified  as  desired.  To  identify  the  substance,  it  is  conveniently 
transformed  into  kreatinin  by  prolonged  boiling  (one-half  to  one 
hour)  with  dilute  hydrochloric  acid.  The  resulting  material  is  then 
examined  as  described  in  the  section  on  the  Urine  (page  263). 

In  addition  to  the  common  kreatins  which  have  just  been  con- 
sidered, Gautier  succeeded  in  further  isolating  xanthokreatinin, 
crusokreatinin  and  amphykreatin  from  muscle-tissue.  The  sub- 
stances are,  however,  found  only  in  traces  and  have  not  as  yet  been 
studied  in  detail. 

THE  XANTHIN-BASES. 

The  xanthin-bases  which  have  been  isolated  from  muscle-tissue 
comprise  xanthin,  hypoxanthin,  and  guanin.  In  addition,  an- 
other basic  substance  has  been  obtained,  which  is  termed  carnin. 
This  is  manifestly  closely  related  to  the  xan thins  proper,  as  on 
oxidation  it  is  transformed  into  hypoxanthin.  These  bodies  are 
formed  during  the  metabolism  of  the  muscle  nuclei,  and  are  in  part 
eliminated  in  the  urine  as  sucli.  A  variable  fraction,  however,  is 
directly  oxidized  to  uric  acid,  which  in  turn  may  contril)ute  to  the 
formation  of  urea,  but  is  in  part  also  eliminated  directly  (see  page 
259). 


THE  XANTHIN-BASES.  391 

Isolation. — To  demonstrate  the  presence  of  the  xanthin-bases  in 
muscle-tissue  it  is  necessary  to  work  with  large  amounts  of  material, 
as  the  quantity  present  is  always  small.  On  the  whole  it  is  more 
convenient  to  start  with  some  extract  of  beef,  such  as  that  of  Liebig, 
and  to  proceed  as  follows  : 

The  material  in  question  is  taken  up  with  an  amount  of  water 
which  is  just  sufficient  for  its  solution,  at  a  temperature  of  about 
50°  C.  The  liquid  is  then  precipitated  with  a  solution  of  sub- 
acetate  of  lead.  The  resulting  filtrate  we  term  A.  The  pre- 
cii)itate  contains  the  carnin  in  combination  with  lead.  To  isolate 
the  substance  the  mass  is  suspended  in  water  and  boiled.  The 
filtrate  while  still  hot  is  saturated  with  hydrogen  sulphide,  which 
decomposes  the  lead  compound  of  carnin  with  the  liberation  of  the 
free  base.  The  lead  sulphide  is  filtered  off,  the  filtrate  concen- 
trated to  a  small  volume  and  precipitated  with  a  concentrated 
solution  of  silver  nitrate.  *  Ammonia  is  added  to  dissolve  any  pre- 
cipitated chlorides  when  the  insoluble  silver-carnin  is  placed  in 
a  small  amount  of  hot  water  and  is  decomposed  with  hydrogen 
sulphide.  The  solution  is  filtered  while  hot,  decolorized  Avith 
animal  charcoal,  and  precipitated  with  alcohol.  The  carnin  is  thus 
thrown  down. 

Filtrate  A  is  decomposed  with  hydrogen  sulphide,  filtered  and 
strongly  concentrated,  when  on  standing  kreatin  separates  out. 
The  filtrate  is  rendered  alkaline  with  ammonia  and  is  precipitated 
with  ammoniacal  silver  nitrate  solution.  The  xanthin  bases  are  thus 
thrown  down  as  double  silver  salts.  They  are  filtered  off  and  dis- 
solved in  a  small  amount  of  hot  nitric  acid.  The  solution  is  placed 
in  the  refrigerator  when  the  salts  of  hypoxanthin  and  guanin  crystal- 
lize out.     The  filtrate  is  termed  B. 

To  separate  the  guanin  from  the  hypoxanthin,  the  material  is  sus- 
pended in  water ;  the  liquid  is  brought  to  the  boiling-point  and 
treated  with  a  solution  of  ammonium  sulphide,  drop  by  drop.  The 
silver  salts  are  thus  decomposed.  The  liquid  is  filtered  while 
hot.  The  filtrate  C  contains  a  portion  of  the  guanin  and  all  of 
the  hypoxanthin,  while  a  fraction  of  the  guanin  remains  in  the 
precipitate.  This  can  be  extracted  by  boiling  with  a  very  dilute 
solution  of  hydrochloric  acid,  when  the  free  base  is  precipitated 
by  adding  an  excess  of  ammonia  to  the  acid  solution.  Filtrate  C 
is  placed  on  a  water-batli  and  treated  with  ammonia,  when  the 
remaining  portion  of  the  guanin  is  thrown  down.  The  hypoxanthin 
is  then  obtained  after  filtration  on  evaporating  the  ammoniacal 
solution. 

Filtrate  B  contains  the  xanthin  salt  of  silver  nitrate.  To  isolate 
the  free  base  the  double  salt  is  first  precipitated  from  its  acid  solu- 
tion by  ammonia.  It  is  suspended  in  water,  decomposed  with 
hydrogen  sulphide,  and  extracted  with  ammonia.  On  evaporation 
the  xanthin  crystallizes  out. 

This  method  is  well  adapted  for  extracting  the   xanthin   bases 


392  THE  MUSCLE-TISSUE. 

from  any  organs  of  the  body,  and  also  serves  for  the  isolation  of 
adenin.  Should  this  be  present,  it  is  found  in  filtrate  C.  On  adding 
ammonia  the  adenin  together  with  the  hypoxanthin  remains  in  solu- 
tion. On  cooling  the  adenin  separates  out,  especially  after  the 
ammonia  has  been  evaporated  oif.  Hypoxanthin  remains  in  solu- 
tion and  is  obtained  on  final  evaporation. 

As  the  general  chemical  relations  of  the  xanthin-bases  have 
already  been  considered  (page  101),  it  will  suffice  to  give  a  brief 
account  of  the  more  important  properties  of  the  individual  sub- 
stances at  this  place. 

Xanthin. — Purexanthin  is  either  amorphous  or  occurs  in  the  form 
of  fine  platelets  which  are  gathered  into  little  clumps  so  that  the 
material  often  presents  a  granular  aspect.  In  cold  water  it  is  almost 
insoluble,  and  in  hot  water  also  it  dissolves  with  difficulty.  In 
alcohol  and  ether  it  is  insoluble.  In  acids  and  alkalies  it  dissolves 
Avith  comparative  ease,  but  at  the  same  tnne  it  combines  with  these 
to  form  compounds,  which  are  for  the  most  part  readily  crystalliz- 
able.  From  its  ammoniacal  solution  it  is  obtained  as  such  on  evapo- 
ration of  the  ammonia.  .  From  this  solution  it  is  thrown  down 
by  silver  nitrate  as  a  gelatinous  precipitate  of  the  composition 
CgH^NjO^.Ag^O.  If  this  is  dissolved  in  nitric  acid,  a  double  salt 
results,  which  crystallizes  out  on  standing.  Unlike  hypoxanthin, 
xanthin  is  precipitated  by  an  ammoniacal  solution  of  lead  sub- 
acetate.  From  its  aqueous  solution  it  is  precipitated  as  a  greenish- 
yellow  material  by  means  of  cupric  acetate  on  boiling. 

Tests. — Nitric  Acid  Test. — On  evaporating  a  few  crystals  of 
xanthin  on  platinum  foil  with  nitric  acid  a  yellow  spot  remains, 
which  turns  red  when  moistened  with  a  drop  of  sodium  hydrate 
solution.  On  further  heating,  it  becames  a  beautiful  purplish  violet. 
The  reaction  is  thus  similar  to  the  murexid  test  for  uric  acid,  but  it 
will  be  noted  that  in  this  case  a  red  color  develops  on  the  addition 
of  the  alkali,  while  with  uric  acid  a  blue  color  is  obtained. 

Hoppe-Seyler's  Test. — A  few  crystals  of  xanthin  are  placed 
in  a  watch-crystal  containing  a  mixture  of  a  few  drops  of  a  solution 
of  sodium  hydrate  and  of  calcium  hypochlorite.  A  dark -green  zone 
then  appears  about  the  xanthin,  which  subsequently  turns  brov.n 
and  ultimately  disappears. 

Weidel's  Test. — A  small  amount  of  xanthin  is  cov^ered  with 
freshly  prepared  chlorine-water  containing  a  trace  of  nitric  acid.  The 
mixture  is  evaporated  to  dryness  on  a  water-bath,  and  the  residue 
exposed  to  the  fumes  of  ammonia,  when  a  beautiful  red  or  purplish- 
violet  color  develops. 

Hypoxanthin  (Sarcin). — Hypoxanthin  crystallizes  in  small  white 
needles.  It  is  soluble  in  hot  water,  less  readily  so  in  cold  water, 
while  in  cold  alcohol  it  is  almost  insoluble.  Like  xanthin,  it  dis- 
solves with  comparative  ease  in  dilate  solutions  of  the  alkaline 
hydrates  and  acids,  forming  salts,  which  are  decomposed  by  distilled 
water,  with  the  liberation  of  the  free  base.    Of  these,  the  chlorhydrate 


THE  XANTHIN-BASES.  393 

is  fairly  characteristic,  as  on  rapid  evaporation  it  crystallizes  out  in 
distinct  whetstone  crystals  similar  to  those  of  uric  acid.  On  treat- 
ing the  substance  in  aninioniacal  solution  with  an  animoniacal 
solution  of  silver  nitrate,  a  double  salt  of  hypoxanthin  with  silver 
is  precipitated,  which  is  soluble  with  difficulty  in  boiling  nitric  acid. 
On  cooling,  it  separates  out  in  the  form  of  curiously  bent  prisms, 
which  are  quite  characteristic.  On  boiling  with  a  solution  of  acetate 
of  copper,  hypoxanthin  is  thrown  down  as  a  cupric  salt. 

Test. — The  substance  does  not  give  the  common  reactions  of 
xanthin.  When  treated  with  zinc  and  hydrochloric  acid,  however, 
hypoxanthin  gives  rise  to  a  ruby-red  color,  which  later  changes  to  a 
brownish  red,  when  sodium  hydrate  solution  is  added  in  excess. 

Guanin. — Guanin  is  usually  obtained  in  amorphous  form,  but  can 
be  brought  to  crystallize  out  from  its  solutions  in  strong  ammonia, 
on  spontaneous  evaporation.  It  is  insoluble  in  water,  alcohol,  and 
ether.  In  mineral  acids  it  dissolves  with  comparative  ease,  at  the 
same  time  forming  salt-like  products,  which  are  crystallizable,  but 
quite  unstable,  so  that  in  the  case  of  some  of  them  at  least  the  free 
base  is  liberated  by  water.  With  the  common  alkalies  it  likewise 
combines  to  form  compounds  which  are  somewhat  soluble  in  warm 
water,  but  more  readily  so  if  a  little  fixed  alkali  is  present.  In 
ammonia  it  dissolves  with  great  difficulty,  so  that  it  is  possible  to 
precipitate  the  substance  from  its  acid  solutions  by  the  addition  of 
ammonia.  Silver  nitrate  precipitates  the  substance  from  its  solu- 
tions in  nitric  acid  as  a  double  salt,  which  dissolves  in  boiling  nitric 
acid,  and  crystallizes  out  on  cooling  in  the  form  of  fine  needles.  On 
boiling  a  solution  of  guanin  (as  calcium  compound)  with  acetate  of 
copper  the  corresponding  salt  is  thrown  down. 

Tests. — Like  xanthin,  guanin  gives  the  nitric  acid  reaction,  but 
with  a  somewhat  more  bluish-violet  color,  while  Hoppe-Seyler's  test 
and  that  of  Weidel  are  negative.  With  picric  acid  it  combines  to 
form  a  yellow  crystalline  precipitate  when  a  saturated  solution  of 
the  acid  is  added  to  a  warm  solution  of  the  hydrochlorate. 

Adenin. — Adenin  crystallizes  with  three  molecules  of  water  in  the 
form  of  long  hexagonal  needles.  If  these  are  placed  in  an  amount 
of  water  which  is  insufficient  for  their  solution  and  heat  is  now 
applied  to  the  temperature  of  53°  C,  they  suddenly  lose  their  trans- 
parency and  become  opaque.  This  peculiar  behavior  may  aid  in  the 
identification  of  the  substance.  It  is  soluble  with  difficulty  in  cold 
Avater,  more  readily  so  in  hot  water,  but  is  insoluble  in  ether.  In 
hot  alcohol  it  dissolves  to  a  slight  extent.  In  acids  and  alkalies  it 
is  soluble  with  ease.  In  ammonia  it  is  less  readily  soluble  than 
hypoxanthin,  but  more  readily  so  than  guanin.  From  its  alkaline 
solutions  the  free  base  is  precipitated  by  the  addition  of  very  dilute 
acids,  care  being  taken  to  avoid  an  excess.  With  silver  nitrate  it 
forms  a  compound  which  is  soluble  with  difficulty  in  boiling  nitric 
acid,  and   crystallizes  out  on  cooling.     Like  the  xanthin  bases,  that 


394  THE  MUSCLE-TISSUE. 

have  already  been  considered,  it  is  precipitated  from  its  solutions  on 
boiling  witli  acetate  of  copper  as  a  double  salt. 

Tests. — Like  hypoxanthin,  it  does  not  give  the  common  xanthin 
reactions,  nor  does  it  react  with  zinc  and  hydrochloric  acid.  It  is 
most  readily  identified  by  the  behavior  of  its  crystals,  as  has  just 
been  described,  or  Iw  adding  a  solution  of  auric  chloride  to  its 
solution  as  a  hydrochlorate,  when  a  double  salt  is  formed,  which 
crystallizes  in  part  in  octahedral  or  prismatic  crystals,  the  angles  of 
which  are  often  rounded  off. 

Carnin. — Carnin,  as  I  have  stated  before,  is  not  a  true  xanthin- 
base,  but  is  manifestly  closely  related  to  this  group,  as  on  oxidation 
with  bromine-water  or  with  nitric  acid  it  is  transformed  into  hypo- 
xanthin. 

It  is  obtained  in  indistinctly  crystalline  form.  In  hot  water  it  is 
readily  soluble,  while  in  cold  Avater  it  dissolves  with  great  difficulty, 
and  is  insoluble  in  alcohol  and  ether.  It  is  neutral  in  reaction,  and 
combines  with  acids  and  alkalies  to  form  salts.  These  salts  are  not 
decomposed  by  water.  Its  hydrochlorate,  which  results  whenadenin 
is  dissolved  in  warm  hydrochloric  acid,  crystallizes  out  on  cooling, 
and  combines  with  platinum  chloride  to  form  a  double  salt.  Silver 
nitrate  precipitates  it  from  its  aqueous  solutions  as  a  silver  salt,  and 
it  is  to  be  noted  that  this  compound  is  insoluble  both  in  ammonia 
and  nitric  acid.  Subacetate  of  lead  precipitates  adenin  as  a  lead 
salt ;  this  is  soluble  in  l^oilin^  water.  Acetate  of  copper  produces 
no  precipitate.  The  substance  gives  none  of  the  common  reactions 
of  the  xanthin-bases,  and  is  best  identified  by  the  behavior  of  its 
lead  and  silver  salts. 

Still  other  nitrogenous  extractives  may  be  obtained  from  muscle- 
tissue,  but,  with  the  exception  of  taurin,  inosinic  acid,  and  carnosin 
they  are  scarcely  known.     For  a  description  of  taurin  see  page  170. 

Inosinic  Acid. — Inosinic  acid  is  apparently  a  constant  con- 
stituent of  muscle-tissue,  but  is  most  abundantly  encountered  in  the 
muscles  of  ducks,  from  which  Creite  was  able  to  isolate  as  much  as 
0.26  per  cent.,  calculated  as  barium  salt. 

The  substance  has  the  composition  CioIIj3N^PO^,  and  is  com- 
monly regarded  as  a  nucleinic  acid.  On  decomposition  with  boiling 
water  it  is  said  to  yield  hypoxanthin,  trioxy-valerianic  acid,  and 
phosphoric  acid.  Whether  or  not  a  relationship  exists  between 
inosinic  acid  and  phospho-carnic  acid  is  unknown. 

Carnosin. — Carnosin  is  a  basic  substance  which  has  been  found 
in  beef  extract,  and  which  possibly  also  exists  as  such  in  the  fresh 
muscle-tissue.  Its  formula  is  given  by  Gulewitsch  as  C9II14N4O3. 
In  its  general  properties  the  substance  resembles  arginin. 


FAT.  395 


GASES. 

Both,  when  at  rest  as  also  during  its  activity,  the  muscle-tissue  is 
constantly  taking  up  oxygen  from  the  blood  and  the  lymph.  This 
is  stored  in  the  cells  proper,  and  is  extensively  utilized  in  the  oxida- 
tion-processes which  are  constantly  going  on,  but  which  occur  with 
increased  intensity  when  the  muscle  is  at  work.  Carbon  dioxide  is 
similarly  given  off,  and  it  can  be  readily  proved  that  the  oxygen 
which  is  utilized  in  its  formation  is  in  part  at  least  stored  within  the 
tissue.  Carbon  dioxide  is  thus  still  given  off,  even  when  a  muscle 
is  removed  from  the  body  and  worked  in  an  atmosphere  which  is 
free  from  oxygen.  It  has  been  noted,  moreover,  that  the  amount 
which  is  then  set  free  is  the  same  as  that  wliich  results  when  the 
muscle  is  worked  in  the  ]>resence  of  an  abundance  of  oxygen.  Of 
the  form,  however,  in  which  the  gas  exists  in  the  muscle  we  know 
nothing,  but  it  is  manifestly  not  present  in  the  free  state,  as  no 
oxygen  at  all,  or  very  small  amounts  only,  can  be  extracted  by  a 
vacuum  pump.  With  increasing  activity  larger  amounts  of  oxygen 
are  taken  up,  while  larger  amounts  of  carbon  dioxide  are  being 
given  off.  This  difference  is  well  shown  in  the  following  table, 
which  is  taken  from  Gautier.  The  figures  have  reference  to  100 
volumes  of  blood,  calculated  at  0°  C.  and  1000  Hgmm.  pressure: 

Carbon 
Oxygen,      dioxide. 

Arterial  blood  from  muscle-tissue 15.25  26.71 

Venous  blood  from  muscle-tissue  while  at  rest  .    .       6.70  33.20 

Venous  blood  from  muscle-tissue  while  at  work    .    .       2.97  36.38 

In  addition  to  carbonic  acid,  small  amounts  of  nitrogen  can  fur- 
ther be  obtained  from  muscle-tissue,  which  are  manifestly  absorbed 
from  the  blood  and  apparently  exist  in  a  state  of  solution.  As  in 
other  tissues  and  fluids  of  the  body  where  nitrogen  is  also  found,  its 
presence  is  probably  of  no  significance.  The  quantity  that  can  be 
obtained  by  the  vacuum  pump  is  essentially  the  same  as  that  which 
is  found  in  the  lymph  and  in  the  blood. 

FAT. 

The  amount  of  fat  which  is  found  in  muscle-tissue  varies  not  only 
with  different  animals  but  also  in  one  and  the  same  individual  at 
different  periods  of  life.  Some  of  the  analytical  results  which  have 
been  obtained  are  shown  below  : 

Pro  mille. 

Lean  beef 6.1-7.6 

Eabbit       10.7 

Partridge 14.3 

Pig 40.0-90.0 

Salmon      100.0 

Mackerel      164.0 

Eel 329.0 


396  THE  MUSCLE-TISSUE. 

The  fat  is  deposited  not  only  in  the  interfibrillary  connective 
tissue,  but  also  in  the  sarcoplasra  proper,  and  is  apparently  more 
abundant  in  the  red  meat,  which  contains  more  sarcoplasm  than  in 
white  meat. 

Like  glycogen,  it  here  represents  a  reserve  source  of  muscular 
energy,  but  is  apparently  utilized  more  especially  when  a  sufficient 
su])ply  of  the  former  or  of  grape-sugar,  as  such,  is  not  available. 

While  it  is  ordinarily  derived  from  the  ingested  fats  or  from  carbo- 
hydrates, there  can  be  no  doubt  that  under  certain  pathological  condi- 
tions, which  are  associated  with  an  increased  destruction  of  tissue 
albumins,  it  can  also  originate  from  these.  This  question,  however, 
we  shall  not  consider  in  detail  at  this  place,  but  shall  revert  to  it  in 
a  future  section. 

In  addition  to  fats,  muscle-tissue  also  contains  a  small  amount  of 
cholesterin,  fats,  and  fatty  acids,  and  at  times  considerable  quanti- 
ties of  lecithins  (0.69  per  cent.). 

The  chemical  composition  of  involuntary  muscle-tissue  is  appar- 
ently similar  to  that  of  the  striped  variety.  From  the  muscles  of 
the  stomach  of  the  pig  and  goose  Velichi  obtained  a  spontaneously 
coagulating  plasma,  from  which  one  albumin  was  precipitated  on 
dialysis,  while  a  second  remained  in  solution.  The  first  was  solu- 
ble in  dilute  solutions  of  the  neutral  salts  and  was  precipitated  by 
acetic  acid ;  it  coagulated  between  54°  and  60°  C.  The  second 
coagulated  between  46°  and  50°  C 

In  the  holothurians,  which  probably  represent  one  of  the  lowest 
forms  of  animal  life  in  which  an  actual  differentiation  of  proto- 
plasm to  muscle-tissue  has  occurred,  the  tissue  is  apparently  of  the 
unstriped  variety.  In  stictopus,  which  belongs  to  this  order, 
v.  Fiirth  was  unable  to  isolate  an  albumin  coagulating  below  50°  C, 
while  Krukenberg  states  that  in  the  case  of  Holothuria  tubulosa  his 
extraction  liquid  coagulated  at  45°  C. 


CHAPTER  XVII. 


THE  NERVE-TISSUE. 


Owing  to  the  difficulty  which  attends  the  separation  of  the  vari- 
ous morphological  components  of  nerve-tissue,  and  the  peculiar  prop- 
erties of  some  of  its  most  important  chemical  constituents,  it  is  as 
yet  impossible  to  give  an  account  of  its  chemical  composition  which 
is  at  all  satisfactory.  Here,  as  in  the  other  organs  of  the  body,  we 
meet  with  certain  substances  which  are  generally  spoken  of  as  ex- 
tractives, and  which  are  manifestly  katabolic  products  that  result 
during  the  functional  activity  of  the  tissue  in  question.  These  are 
in  no  sense  specific  of  nerve-tissue,  however.  They  comprise  kreatin, 
uric  acid,  urea,  xanthin,  hypoxanthin,  guanin,  adenin,  inosit,  and 
lactic  acid — viz.,  substances  which,  as  we  have  just  seen,  are  also 
found  in  muscle-tissue.  In  addition,  we  find  certain  albumins 
which  in  part  belong  to  the  native  albumins  and  in  part  to  the  globu- 
lins ;  further,  nucleins  and  albuminoids,  among  which  the  so-called 
neurokeratin  is  of  special  interest,  as  it  largely  enters  into  the  com- 
position of  the  supporting  structure  of  the  nerve-tissue,  and  is  char- 
acteristic of  this.  Besides  these  various  substances  nerve-tissue 
contains  especially  large  amounts  of  so-called  myelins — viz.,  lecithin, 
cholesterin,  and  protagon.  A  certain  amount  of  mineral  salts  and 
a  large  quantity  of  water  constitute  the  remaining  known  components 
of  the  tissue. 

As  regards  the  distribution  of  these  substances  among  the  gangli- 
onic cells  and  nerve-fibres  more  especially,  our  knowledge  is  as  yet 
quite  incomplete ;  but  it  appears  from  the  analyses  which  are  avail- 
able that  the  gray  substance  of  the  brain  in  the  dried  state  consists 
to  the  extent  of  one-half  at  least  of  albumins,  while  the  substances 
which  are  soluble  in  ether  amount  to  only  about  one-quarter  of  the 
total  quantity.  Protagon  is  here  present  in  only  very  small  quantity. 
In  the  white  substance  of  the  brain,  on  the  other  hand,  it  is  found 
in  considerable  amount,  while  the  albumins  constitute  only  one- 
quarter  of  the  dry  material.  In  embryonic  brains,  in  which  the 
medullary  sheaths  are  not  as  yet  developed,  much  smaller  amounts 
of  lecithins  are  found  than  in  the  adult  brain  ;  and  it  is  noteworthy, 
moreover,  that  protagon  and  neurokeratin  are  here  both  absent.  It 
may  thus  be  concluded  that  these  substances  are  essentially  com- 
ponents of  the  medullary  nerve-fibres.  According  to  Hoppe-Seyler, 
indeed,  the  small  amount  of  ]irotagon  which  is  found  in  the  gray 
substance  of   the  brain  is  entirely  referable  to  this  source.     The 

397 


398  THE  NERVE-TISSUK 

presence  of  smaller  amounts  of  lecithins  in  the  embryonic  brain, 
as  compared  with  the  adult  brain,  and  notably  the  gray  matter,  is 
now  generally  explained  upon  the  assumption  that  the  material 
in  question  is  in  some  manner  intimately  concerned  in  the  growth 
of  the  cellular  elements  proper. 

Analysis  of  Brain-tissue  (Baumstark). 

White  nmtter.i    Gray  matter.^ 

Water 69.53  77.00 

Solids 30.47  23.00 

Insoluble  albumins  and  connective-tissue    .    .       5.00  6.08 

Neurokeratin 1.89  1.04 

Nucleins 0.29  0.20 

Protagon 2.51  1.08 

Cholesterins,  free 1.82  0.63 

Cholesterins,  combined 2.69  1.75 

Mineral  salts .       0.52  0.56 

Other  substances,  soluble  in  ether 30.47  23.00 

Analysis  of  the  Mineral  Salts  (Geoghegan). 

Chlorine 0.42  —  1.06 

Phosphoric  acid  (PO J 0.85—1.39 

Carbonic  acid  (CO3) 0.25  —  0.33 

Sulphuric  acid  (SOJ 0.14  —  0.13 

Phosphate  of  iron  (Fe2(P04)j) 0.09  —  0.30 

Calcium 0.02 

Magnesium 0.06  —  0.07 

Potassium 0.58  —  1.52 

Sodium 0.45  —  0.78 

In  the  brain  Gautier  has  demonstrated  traces  of  arsenic. 

Albumins. — Of  the  character  of  the  individual  albumins  which 
occur  in  nerve-tissue  very  little  is  known.  Baumstark  states  that 
they  are  essentially  the  same  as  those  of  muscle-tissue,  and,  to  judge 
from  the  researches  of  Petrowski,  it  appears  that  one  of  these  may 
be  identical  with  myosin,  as  it  is  soluble  in  dilute  saline  solution, 
and  can  be  precipitated  by  diluting  with  water  or  by  salting  with 
sodium  chloride.  The  coagulation-point  of  the  substance  has,  how- 
ever, not  been  determined.  It  is  .said  to  occur  both  in  the  gray  and 
the  white  matter. 

More  recently  Halliburton  claims  to  have  isolated  two  neuro- 
globulins,  both  from  the  w^hite  and  the  gray  matter,  with  a  coagu- 
lation-point of  47°  and  75°  C,  respectively.  In  addition  he  found 
a  nucleo-alhnmin  in  the  gray  substance,  with  0.5  per  cent,  of  phos- 
phorus, which  coagulated  between  55°  and  60°  C.  v.  Jaksch 
further  claims  to  have  i.solated  a  nuclein  from  the  gray  matter  which 
contains  but  little  pho.sphorus,  and  yields  hypoxanthin,  xanthin, 
phosphoric  acid,  and  an  albuminous  body  on  decomposition. 

The  albumins,  as  I  have  indicated,  are  principally  found  in  the 
gray  matter  of  the  brain,  and  constitute  about  one-half  of  the 
dried  substance.  In  the  wliite  matter,  however,  they  are" also  found, 
though  in  much  smaller  amount,  and  it  is  thought  that  the  cylinder 

^  The  complete  separation  into  gray  and  white  matter  is,  of  course,  impossible. 


THE  NERVE-TISSUE.  399 

of  the  nerve-fibre  is  essentially  of  an  albuminous  nature.  They  are 
without  doubt  intimately  concerned  in  the  specific  function  of  the 
nerve-tissue,  but  of  the  part  which  they  take  in  such  function  noth- 
ing whatever  is  known.  It  is  interesting  to  note,  however,  that 
the  gray  matter  of  the  brain,  as  also  of  the  spinal  cord  and  groups 
of  ganglionic  cells  outside  of  the  central  nervous  system,  always  pre- 
sent an  acid  reaction,  ^vhile  the  white  matter  of  the  brain  and  cord 
and  the  peripheral  nerves  is  always  neutral  or  slightly  alkaline. 
The  substance  which  produces  the  acid  reaction  of  the  gray  matter 
is  apparently  the  common,  optically  inactive  lactic  acid,  and  it  is 
noteworthy  that  in  the  nerve-tissue  also  a  lactic  acid  is  encountered 
in  those  portions  which  are  especially  rich  in  albumins.  But  while 
the  acid  reaction  of  muscle-tissue  becomes  manifest  only  after  death, 
it  can  be  readily  shown  tliat  in  the  case  of  the  brain  and  spinal  cord 
this  is  normal  even  during  life.  Whether  or  not  other  substances 
besides  lactic  acid  contribute  to  the  acid  reaction  of  the  gray  matter 
has  not  been  definitely  established.  But  it  is  quite  likely  that 
this  is  the  case,  as  in  the  presence  of  lactic  acid  a  transformation  of 
diphosphates  to  monophosphates  would  of  necessity  occur.  Bibra 
and  Miiller,  moreover,  claim  to  have  obtained  traces  of  formic  acid 
from  the  aqueous  extract  of  the  gray  matter.  Paralactic  acid  has 
not  been  found  in  nerve-tissue. 

Neurokeratin. — This  substance,  which  was  first  isolated  by 
Kiihne,  forms  the  greater  portion  of  the  supporting  tissue  of  the 
central  nervous  system,  and  is  likewise  found  in  the  medullary 
fibres,  where  it  constitutes  the  axilemma  and  outer  sheath  of  the 
medullary  substance.  According  to  some  observers,  moreover,  it 
forms  a  fine  reticulated  network  in  the  latter. 

Neurokeratin  is  an  albuminoid  and  belongs  to  the  group  of  the 
keratins,  which  are  found  widely  distributed  among  the  tissues  of 
epiblastic  origin.  In  the  invertebrate  animals,  in  which  medullary 
fibres  are  not  found,  and  chitinous  substances  largely  enter  into 
the  composition  of  the  outer  skeleton  of  the  body,  it  is  accordingly 
represented  by  a  neurochitin. 

Neurokeratin  is  insoluble  in  water,  ether,  alcohol,  in  dilute  solu- 
tions of  the  alkaline  hydrates,  in  gastric  juice  and  pancreatic  juice. 
To  isolate  the  substance  from  nerve-tissue,  this  is  accordingly  ex- 
tracted with  alcohol  and  ether,  to  remove  the  myelin  substances. 
The  remaining  material  is  freed  from  albumins  and  other  albuminoids 
by  digestion  with  gastric  juice,  and  is  then  treated  with  a  dilute  solu- 
tion of  sodium  hydrate,  which  dissolves  the  nucleins.  The  keratin 
then  remains.  From  the  other  keratins,  which  may  be  obtained 
from  hair,  nails,  horns,  etc.,  neurokeratin  differs  especially  in  its  rela- 
tively small  amount  of  sulphur,  and  the  large  amount  of  carbon  and 
hydrogen  and  the  smaller  quantity  of  nitrogen  which  it  contains. 
This  is  shown  in  the  following  table,  which  is  taken  from  Ham- 
marsten  : 


400  THE  NERVE-TISSUE. 

Carbon.  Hydrogen.  Nitrogen.  Oxygen.  Sulphur. 

Human  hair   ....  50.Bd               6.36  ll.U  20.85         5.00 

Nails         51.00               6.94  17.51  21.85         2.80 

Horn         50.86               6.94  .    .  .    .           3.30 

Egg-shell 49.78               6.56  16.43  22.90        4.25 

Turtle  shell      ....  54.89               6.94  16.77  19.56         2.22 

Neurokeratin  ....  56.11-58.45  7.26-9.02  11.50-14.32    .    .  1.63-2.24 

Its  properties  are  the  same  as  those  of  the  keratins  iu  general 
(which  see). 

THE   MYELIN   BODIES. 

In  former  years  it  was  supposed  that  the  medullary  substance 
of  nerve-fibres  consisted  of  a  single  substance,  myelin,  which  was 
characterized  by  the  fact  that  on  treating  with  water  it  formed 
double-contoured  droplets,  which  can  readily  be  seen  on  microscopical 
examination.  According  to  Gad  and  Heymans,  this  myelin  is  in 
reality  lecithin  in  the  free  state  or  in  loose  combination,  and  we  know 
as  a  matter  of  fact  that  the  peculiar  reaction  is  due  to  decomposition- 
products  of  such  complex  substances  as  protagon  and  certain  com- 
pound cholesterins.  In  speaking  of  myelin  substances  at  the  present 
time  we  have  reference  to  protagons,  lecithins,  and  cholesterins. 

Protagon. — AVhile  there  is  evidence  to  sliow  that  different  pro- 
tagons exist,  we  are  not  as  yet  in  a  position  to  characterize  such 
forms  individually,  and  for  convenience'  sake  we  shall  speak  of 
protagons  as  a  chemical  unity  at  this  j)lace.  The  substance  is  not 
strictly  characteristic  of  nerve-tissue,  as  it  has  also  been  found  in 
other  organs  of  the  body,  such  as  the  spleen,  in  the  stroma  of  the 
red  cor])uscles,  in  pus,  and  in  spermatozoa.  But  Avhile  it  is  here 
present  in  only  small  amounts,  it  enters  into  the  composition  of 
nerve-tissue  to  a  considerable  extent,  and  is  thus  quantitatively  at 
least  peculiar  to  these  structures.  Whether  or  not  the  substance 
occurs  also  in  the  gray  matter  appears  doubtful,  but  in  the  white 
matter  and  in  the  peripheral  medullated  nerve-fibres  it  is  abundant. 

According  to  Liebreich,  who  was  the  first  to  isolate  protagon 
from  brain-tissue,  the  substance  has  the  composition  Cji6Ho4,N4P02. 
It  is  to  be  noted,  however,  that  the  elementary  analysis  of  different 
preparations  has  given  rise  to  different  results,  which  in  itself  sug- 
gests the  ])robability  that  diflFerent  forms  exist.  According  to  some 
observers,  it  also  contains  sulphur  in  molecular  combination,  but 
recent  investigations  have  shown  that  this  is  probably  not  the  case. 
Worner  and  Thierfelder  have  recently  succeeded  in  obtaining  a  sub- 
stance from  a  solution  of  so-called  protagon,  which  they  term  cere- 
bron.  It  was  free  from  phosphorus,  sulphur,  and  a.sh,  and  gave  the 
following  values  on  elementary  analysis:  C=  69.16,  11=11.54, 
N=1.76.  On  boiling  with  mineral  acids  they  obtained  a  sugar 
(galactose)  and  an  acid,  as  also  an  alkaline  complex  which  has  not 
been  identified. 

On  decomposition  with  boiling  baryta-water  protagon  yields  fatty 
acids,  glycerin-phosphoric  acid,  cholin,  and  in  addition  cerebrosides. 


THE  MYELIN  BODIES.  401 

of  which  three  are  now  recognized.  These  are  known  as  cerebrin, 
kerasin,  or  horaocerebrin,  and  encephalin.  Others  also  may  possibly 
exist,  and  it  is  likely  that  the  pyosin  and  pyogenin,  which  Kossel 
and  Freitag  obtained  from  pus,  belong  to  this  order. 

On  boiling  with  dilate  mineral  acids  protagon  also  yields  a  re- 
ducing substance,  which  is  commonly  regarded  as  galactose,  and  is 
referable  to  the  decomposition  of  the  glucosides  just  mentioned. 

Protagon  is  easily  soluble  in  warm  alcohol  and  ether,  while  in 
cold  alcohol  and  cold  ether  it  dissolves  with  difficulty.  On  cooling 
the  substance  crystallizes  out  in  fine  needles  or  in  waxy  masses, 
which  can  readily  be  broken  up  into  a  fine  po\vder.  On  heating  its 
alcoholic  solutions  to  a  temperature  of  48°  C.  or  on  boiling  its 
ethereal  solutions  the  substance  is  readily  decomposed  into  its  com- 
ponents, as  indicated  above.  In  the  dry  state  it  can  be  heated  to 
a  higher  temperature,  but  it  is  then  also  decomposed  before  100°  C. 
is  reached.  The  resulting  products  melt  between  200°  and  203°  C, 
and  begin  to  volatilize  at  220°.  When  moistened  with  water,  the 
substance  swells  and  is  partly  decomposed,  with  the  formation  of 
so-called  "  myelin "  droplets.  If  much  water  is  added,  an  opaque 
fluid  is  obtained. 

Isolation. — To  isolate  protagon  from  brain-tissue  this  should  be  as 
fresh  as  possible,  as  otherwise  partial  decomposition  occurs  spon- 
taneously. The  material  is  freed  from  its  membranes  and  adhering 
blood,  and  is  then  stirred  to  a  ])ulp,  and  extracted  with  85  per  cent, 
alcohol,  at  a  temperature  of  45°  0.,  using  fresh  portions  of  alcohol 
from  time  to  time  until  a  specimen  no  longer  deposits  a  sediment 
when  cooled  to  0°  C.  The  extracts  are  filtered  at  45°  C,  and  sub- 
sequently kept  at  0°  C.  The  resulting  precipitates  are  extracted 
with  cold  ether  to  remove  cholesterins  and  lecithins,  when  the 
remaining  material  is  pressed  between  filter  paper  and  dried  over 
sulphuric  acid.  It  is  finally  pulverized,  again  extracted  with  alcohol 
at  45°  C,  when  the  solution  is  filtered  and  cooled  to  0°  C.  To 
purify  the  substance  it  is  recrystallized  from  warm  alcohol  or  ether. 

Cerebrin. — Cerebrin,  as  I  have  stated,  is  a  normal  decomposition- 
product  of  protagon,  but  probably  does  not  occur  in  the  livnng 
nerve-tissue  as  such.  Associated  with  lecithin,  it  is  also  found  in 
the  stroma  of  the  red  corpuscles  of  the  blood,  in  leucocytes,  in  sper- 
matozoa, in  the  spleen,  in  the  yolk  of  birds'  eggs,  etc.  It  is 
questionable,  however,  whether  it  actually  exists  in  the  froe% 
state,  and  the  fact  of  its  constant  association  with  lecithin  rather 
suggests  that  here  also  it  is  primarily  present  in  the  protagon 
molecule. 

Cerebrin  is  said  to  have  the  formula  C^oHi^QNoOig.  Elementary 
analysis  has  given  the  following  results :  C,  69.08  per  cent.  ; 
H,  11.47;  N,  2.13;  O,  17.32.  On  decomposition  with  boiling 
mineral  acids  it  yields  a  reducing  substance  which  is  commonly 
regarded  as  galactose.  On  oxidation  with  nitric  acid  or  on  fusion 
with  caustic  alkali  palmitic  acid   or  stearic  acid  is  obtained.     If 

26 


402  THE  NERVE-TISSUE. 

the  substance  is  dissolved  in  concentrated  sulphuric  acid,  the 
solution  gradually  assumes  a  purplish-red  color,  which  changes 
to  violet,  and  finally  to  brown.  On  adding  an  equal  amount  of 
water  and  l)oiling,  a  flocculent  precipitate  appears,  which  is  known 
as  cetylid,  and  is  said  to  have  the  composition  C^B[i2„0-g  or 
(Ci6H3i02)3.[Ci6Hi8(OH)3].  This  substance  supposedly  represents 
about  85  per  cent,  of  the  entire  w^eight  of  the  cerebrin.  It 
is  soluble  in  water,  in  hot  alcohol,  and  especially  in  ether  and 
chloroform.  On  fusion  with  caustic  alkali,  cetylid  yields  methane, 
hydrogen,  and  palmitic  acid. 

Pure  cerebrin  is  a  colorless  substance  which  occurs  in  the  form  of 
a  crystalline  powder,  consisting  of  microscopical  globulites.  It  is 
soluble  in  warm  acetone,  acetic  ether,  benzene,  and  boiling  alcohol, 
but  is  insoluble  in  sulphuric  ether,  even  at  its  boiling-point ;  with 
chloroform  it  forms  an  emulsion.  In  cold  water  it  is  likewise 
insoluble.  In  boiling  water  it  swells  to  a  certain  extent,  like  starch 
paste.  It  melts  at  177°  C,  but  is  decomposed  long  before  with 
the  development  of  a  yellow  or  brownish  color.  Its  reaction  is 
neutral. 

Isolation. — Cerebrin  can  be  obtained  either  from  protagon  after 
the  separation  of  tlie  latter  or  directly  from  the  brain  by  decompos- 
ing its  antecedents  with  baryta-water.  To  this  end,  the  material 
after  being  freed  from  its  membranes  and  blood  is  stirred  with 
baryta-water,  and  brought  to  the  boil.  The  insoluble  portion  is 
then  pressed  out  and  repeatedly  extracted  with  alcohol  by  boiling. 
The  extract  is  filtered  while  still  hot,  when  on  cooling  to  0°  C, 
the  cerebrin  separates  out  in  impure  form.  It  is  freed  from  choles- 
terin  and  fats  hy  skakiug  with  ether,  and  purified  by  repeated  solu- 
tion in  hot  alcohol  and  subsequent  cooling,  until  jelly-like  pre- 
cipitates, which  are  referable  to  homocerebrin  or  encephalin,  are  no 
longer  ol)tained. 

With  this  method,  however,  a  considerable  portion  of  the  cerebrin 
is  decomposed,  and  Kossel  has  accordingly  suggested  that  the 
protagon  be  first  extracted  and  subsequently  decomposed.  To  this 
end,  the  substance  is  dissolved  in  methyl  alcohol,  and  is  treated  with 
a  hot  methyl  alcoholic  solution  of  barium  hydrate.  The  mixture  is 
w^armed  for  a  few  minutes  on  a  water-bath,  and  then  allowed  to 
cool.  The  resulting  precipitate  contains  the  entire  amount  of 
•cerebrin  which  can  be  obtained,  and  re])resents  50  per  cent,  of  the 
original  quantity  of  protagon.  It  is  filtered  off,  suspended  in  water, 
and  freed  from  barium  by  a  current  of  carbon  dioxide.  The  insolu- 
ble residue  contains  the  cerebrosides,  which  are  now  extracted  with 
hot  alcohol  and  isolated  by  fractional  crystallization. 

Homocerebrin  (Kerasin). — The  formula  of  homocerebrin  is 
given  as  CoHi.^X^Oi^,  and  the  substance  could  hence  be  regarded  as 
an  anhydride  of  cerebrin.  In  the  dry  state  it  occurs  as  a  wax-like 
mass,  which  can  be  pulverized  only  with  difficulty.  From  its  solu- 
tion in  alcohol  and  boiling  ether  it  separates  out  in  aggregates  of 


THE  MYELIN  BODIES.  403 

exceedingly  fine  needles.  At  130°  C.  it  is  decomposed  with  the 
appearance  of  a  yellow  color,  and  melts  at  150°  C.  Toward  con- 
centrated sulphuric  acid  and  water  it  behaves  like  cerebrin.  Like 
this,  it  yields  a  reducing  substance  of  the  character  of  galactose 
when  boiled  with  dilute  mineral  acids,  and  gives  rise  to  the  forma- 
tion of  cetylid,  like  cerebrin. 

The  amount  of  homocerebrin  which  may  be  obtained  from  pro- 
tagon  is  about  one-fourth  that  of  cerebrin.  It  is  isolated  in  associa- 
tion with  the  latter,  as  described,  and  can  be  separated  from  the 
cerebrin  by  fractional  crystallization  from  the  alcoholic  solution,  in 
which  it  is  more  readily  soluble  than  cerebrin. 

Encephalin. — Encephaliu  is  regarded  as  a  transformation-product 
of  cerebrin,  and,  like  this,  yields  galactose  on  boiling  with  dilute 
mineral  acids.  Cetylid  is  also  obtained  on  treating  with  concentrated 
sulphuric  acid.  It  is  soluble  in  hot  alcohol,  but  tends  to  separate 
out  on  cooling  as  a  jelly-like  material.  On  slow  evaporation  it 
crystallizes  in  platelets,  which  melt  at  150°  C,  but  are  already 
decomposed  at  125°  C  In  hot  water  it  swells  to  form  a  thick 
paste,  which  remains  on  cooling.  Like  homocerebrin,  the  substance 
is  found  in  the  alcoholic  solution  after  the  cerebrin  has  separated 
out  (see  above),  and  can  be  isolated  by  fractional  crystallization 
from  the  mother-liquor,  or  from  a  solution  of  acetone,  in  which  the 
homocerebrin  is  likewise  soluble. 

Lecithins. — The  lecithins,  which  may  be  isolated  from  nerve- 
tissue,  where  they  largely  exist  in  combination  with  the  cerebrosides, 
in  the  form  of  protagon,  but  undoubtedly  also  occur  as  such  in  the 
free  state,  are  as  yet  but  little  known.  On  decomposition  they 
yield  palmitic  acid,  stearic  acid,  oleic  acid,  glycerin-phosphoric  acid, 
and  cholin.  Of  their  significance  nothing  definite  is  known,  but  it 
appears  that  they  are  in  some  manner  intimately  concerned  in  the 
development  of  the  cells,  and  it  is  for  this  reason  probably  that 
much  smaller  amounts  can  be  obtained  from  the  embryonic  brain 
than  from  that  of  the  adult. 

(For  the  general  description  of  the  lecithins,  see  p.  78). 

Isolation. — To  isolate  the  lecithins  of  the  brain,  the  following 
method,  which  has  been  suggested  by  Ziilzer,  is  conveniently  em- 
ployed. The  brain,  which  must  be  perfectly  fresh,  is  freed  from 
membranes,  cut  into  thin  pieces,  and  placed  in  a  jar  with  ether. 
The  material  should  rest  on  a  layer  of  cotton.  After  standing  for 
several  days  the  ethereal  extract  is  poured  oif,  and  separated  from 
the  lower  layer  of  blood.  The  extraction  is  continued  wdth  new  por- 
tions of  ether  so  long  as  anything  passes  into  solution.  If  desired, 
the  remaining  material  can  then  be  extracted  with  80  per  cent,  alco- 
hol at  45~^  C,  which  takes   up   the  protagon,  as  has  been  described. 

The  ethereal  extracts  are  united  and  concentrated  in  the  vacuum. 
Any  protagon  that  has  passed  into  solution  is  thus  thrown  down 
and  filtered  off.  The  clear  solution  is  now  treated  with  an  excess 
of  acetone  so  long  as   a  precipitate  is  formed.     This  is  filtered  oif 


404  THE  NERVE-TISSUE. 

and  thoroughly  washed  with  acetone.  The  acetone-ethereal  solution 
we  terra  A,  and  the  precipitate  B.  A,  contains  the  entire  quantity 
of  cholesterin.  To  recover  this,  the  acetone-ether  is  distilled  off, 
the  residue  is  boiled  with  alcohol,  the  alcoholic  solution  is  filtered 
while  still  hot,  when,  on  cooling,  the  substance  crj^stallizes  out.  Its 
melting-point  is  145°  C. 

The  precipitate  B  is  placed  in  ether.  This  dissolves  the  greater 
portion,  while  a  smaller  amount  remains  undissolved.  The  latter 
consists  of  protagon,  which  was  previously  held  in  solution,  owing 
to  the  presence  of  cholesterin.  The  soluble  portion  is  treated  with 
alcohol  so  long  as  a  precipitate  forms.  This  precipitate  we  term  C, 
and  the  alcoholic  filtrate  1).  If  a  specimen  of  D  is  treated  with  an 
alcoholic  solution  of  platinum  chloride,  a  precipitate  results  ;  this 
is  not  abundant,  however,  and  consists  of  a  chloroplatinate  of  leci- 
thin. To  isolate  the  lecithins  as  such,  the  alcoholic  solution  is  pre- 
cipitated with  acetone,  or  the  ether-alcohol  is  distilled  off,  when  the 
lecithins  remain  as  a  tough,  wax-like  mass. 

Of  the  nature  of  the  substance  or  substances  which  are  contained 
in  the  precipitate  C,  nothing  definite  is  known.  Ziilzer  apparently 
was  able  to  isolate  one  of  these,  however,  and  found  it  to  contain 
both  nitrogen  and  phosphorus.  He  suggests  that  it  may  possibly 
belong  to  the  so-called  cephalins  of  Thudichum.  But  of  the  nature 
of  these  also  our  knowledge  is  as  yet  insufficient  to  warrant  their 
description  at  this  place. 

The  Cholesterins. — Cholesterins  are  found  in  nerve-tissue,  both 
in  the  free  state,  and  as  so-called  combined  cholesterins,  but  of  the 
chemical  character  of  the  latter  we  are  as  yet  in  ignorance  (see 
p.  80). 

The  isolation  of  free  cholesterin  has  been  described  above. 

The  Extractives. — The  extractives  of  nerve-tissue,  as  I  have 
already  stated,  are  essentially  the  same  as  those  which  can  be 
isolated  from  other  organs  of  the  body.  They  comprise  traces  of 
kreatin,  uric  acid,  xanthin,  hypoxanthin,  guanin,  adenin,  inosit, 
volatile  fatty  acids  (acetic  acid  and  formic  acid),  lactic  acid,  glycogen, 
leucin  (or  rather  a  lower  member  of  the  homologous  series 
Cgnllgn  +  ^O.^,  and  urea.  In  addition,  jecorin,  cholin,  and  neu- 
ridin  have  also  been  found ;  neurin,  on  the  other  hand,  does  not 
occur  in  the  brain  under  normal  conditions.  Very  recently  Panella 
claims  to  have  isolated  phospho-carnic  acid  from  the  normal  brain 
of  dogs,  rabbits,  and  calves. 

Neuridin  is  of  special  interest,  as  the  substance  is  constantly 
formed  during  the  putrefaction  of  meat  and  gelatin,  and  has  also 
been  obtained  from  cultures  of  the  typhoid  organism.  According 
to  Brieger,  who  first  isolated  the  body,  it  is  also  present  in  traces  m 
the  volk  of  birds'  eggs.  It  is  a  diamin  of  the  composition  C^H^Ng. 
With    the  chlorides  of    gold    and    platinum  it  forms  well-defined 


THE  MYELIN  BODIES.  405 

crystalline  salts.     On  boiling  with  caustic  alkalies  it  is  decomposed 
into  trimethyl-amin  and  dimetliyl-amin.     It  is  not  toxic. 

Jecorin  is  a  substance  of  unknown  composition,  but  apparently 
contains  both  sulphur  and  phosphorus.  It  is  not  exclusively  encoun- 
tered in  nerve-tissue,  but  has  also  been  found  in  traces  in  the  liver,  in 
the  muscles,  and,  accordino;  to  some  observers,  in  the  blood.  It 
reduces  cupric  oxide  in  alkaline  solution  on  boiling,  and  on  cool- 
ing separates  out  in  the  form  of  a  thick  jelly.  It  is  soluble  in  ether, 
and  can  be  precipitated  from  its  solutions  by  alcohol. 


CHAPTER    XVIII. 

THE  EYE  AND  THE  EAR. 

THE  EYE. 

In  studying  the  chemical  composition  of  the  eye,  we  shall  con- 
sider the  most  important  parts  of  the  organ  in  succession.  It 
should  be  pointed  out  in  advance,  however,  that  the  subject  has 
thus  far  received  but  little  attention,  and  our  account  must  hence  of 
necessity  be  very  imperfect. 

The  Cornea. — An  analysis  of  the  cornea  of  the  ox  has  given 
the  following  results: 

Pro  mille. 

Water 758.3 

Solids 241.7 

Collagen 203.8 

Other  organic  substances      28.4 

Mineral  salts 9.2 

The  collagen,  which  forms  the  greater  portion  of  the  fibrous  net- 
work of  the  cornea,  is  probably  identical  with  the  common  form 
which  can  be  isolated  from  cartilage,  and,  according  to  Morner,  con- 
tains 16.95  per  cent,  of  nitrogen. 

The  semiliquid  interfibrillary  substance  consists  of  a  mucoid, 
which  yields  a  reducing  substance  on  boiling  with  dilute  mineral 
acids.  It  contains  about  2  per  cent,  of  sulphur,  and  seems  to  be 
characteristic  of  corneal  tissue.  In  addition,  we  find  two  globulins, 
which,  according  to  Morner,  do  not  belong  to  the  cornea  proper, 
however,  but  are  contained  in  the  epithelial  layer.  Nucleins  have 
not  been  found. 

Descemet's  membrane  principally  consists  of  a  membranin,  which 
contains  14.77  per  cent,  of  nitrogen,  and  0.90  per  cent,  of  sulphur. 
The  substance  is  a  glucoproteid,  and  belongs  to  the  group  of  hyalo- 
gens  (which  see).  On  boiling  with  dilute  hydrochloric  acid  it  yields 
a  reducing  substance.  In  ordinary  boiling  water  it  is  insoluble,  but 
dissolves  under  the  action  of  superheated  steam.  It  is  digested 
by  trypsin,  M-hile  the  gastric  juice  is  without  effect. 

The  Sclerotic. — The  composition  of  the  sclerotic  coat  of  the 
eye  is  very  much  the  same  as  that  of  the  cornea,  liut  it  appears  that 
the  quantity  of  the  mucoid  is  here  much  less,  while  collagen  repre- 
sents about  seven-eigliths  of  the  entire  amount  of  solids. 

The  Aqueous  Humor. — The  aqueous  humor  is  a  clear  fluid  of 
an  alkaline  reaction  and  a  specific  gravity  varying  between  1.003  and 
406 


THE  EYE.  407 

1.009.  Its  quantitative  composition  has  already  been  given  (page 
343).  According  to  Griinhagen,  it  contains  traces  of  paralactic  acid, 
a  dextrorotatory  body,  and  a  reducing  substance,  which  is  not  sugar. 
Both  the  latter  are  unknown.  The  albumins  in  question  are  serum- 
albumin,  serum-globulin,  and  traces  of  fibrinogen. 

The  Crystalline  Lens. — The  capsule  of  the  crystalline  lens, 
like  Descemet's  membrane,  consists  essentially  of  a  membranin, 
which  is  not  identical  with  that  found  in  the  latter,  however,  as  it 
is  less  resistant  to  the  action  of  boiling  water  and  of  acids  and  alka- 
lies. According  to  Morner,  it  contains  14.10  per  cent,  of  nitrogen 
and  0.83  per  cent,  of  sulphur. 

A  general  idea  of  the  chemical  composition  of  the  lens  itself  may 
be  formed  from  the  following  analysis,  which  I  have  taken  from 
Neumeister : 

Per  cent. 

Water 63.50 

Solids 36.50 

Albumins 35.00 

Insoluble  albuminoid 17.00 

/3-crystalline J  1.00 

«-crystalline 6.80 

Albumin 0.20 

Fats 0.29 

Lecithins      0.23 

Cholesterin 0.22 

Salts 0.80 

The  albumins  of  the  lens  can  be  divided  into  two  groups,  viz., 
those  which  are  soluble  in  dilute  saline  solution,  and  those  which  are 
insoluble.  The  latter  group  is  represented  by  a  substance  which  is 
spoken  of  as  albumoid.  It  is  manifestly  a  true  albumin,  as  it  is 
entirely  dissolved  by  the  gastric  juice,  and  does  not  yield  a  reducing 
substance  on  boiling  wdth  mineral  acids.  It  gives  all  the  common 
color  reactions  of  the  true  albumins,  and  has  the  same  elementary 
composition.  In  dilute  mineral  acids  and  alkalies  it  dissolves  with 
ease,  and  is  reprecipitated  on  neutralization.  Unlike  the  alkaline 
albuminates,  however,  its  solution  in  dilute  alkalies  coagulates  at 
50°  C,  in  the  presence  of  8  per  cent,  of  sodium  chloride.  The  sub- 
stance manifestly  constitutes  the  greater  portion  of  the  lens-fibres, 
as  the  nitrogen  and  sulphur  values  of  the  two  are  practically  the 
same,  viz.,  N,  16.62  and  S,  0.79  per  cent,  in  the  case  of  the  albumoid, 
as  compared  with  IST,  16.61  and  S,  0.77  of  the  fibres.  It  can  be  shown, 
moreover,  that  after  extraction  of  the  soluble  constituents  of  the 
lens  the  fibrous  framework  remains  and  g-ives  the  same  reactions 
as  the  isolated  albumoid.  Its  amount  increases  from  without 
inward,  in  accordance  with  the  increasing  age  of  the  fibres. 

Aside  from  a  very  small  amount  of  serum-albumin,  the  remaining 
soluble  albumins  of  the  lens  are  represented  by  two  vitellins,  which 
are  termed  a-crystalline  and  ^9-crystalline,  respectively.  Of  these, 
the  a-body  is  notably  found  in  the  outer  portion  of  the  lens, 
while  the  /3-substance  occurs  in  the  inner  portion  more  particularly, 


408  THE  EYE  AND  THE  EAR. 

and  is  apparently  the  only  one  that  is  found  in  the  centre  of  the 
lens. 

The  two  substances  can  be  isolated  from  an  aqueous  extract  of  the 
lens  by  saturating  the  solution  with  magnesium  sulphate  at  a 
temperature  of  30°  C.  The  precipitate  is  then  dissolved  in  water, 
dialyzed,  and  the  resulting  solution  precipitated  with  acetic  acid, 
which  throws  down  the  a-body,  while  the  /5-crystalline  remains  in 
soluti(^n.  A  small  amount  of  the  /5-substance,  it  is  true,  is  also  pre- 
cipitated by  the  acetic  acid,  but  can  be  separated  from  the  a-body  by 
a  repetition  of  the  process. 

Both  substances  are  precipitated  from  their  neutral  solutions  by 
carbon  dioxide,  but  in  the  case  of  the  ,5-crystalline  this  precipitation 
is  never  complete.  The  latter  coagulates  at  63°  C,  and  tlie  a-crys- 
talline  at  72°  C.  The  ^-substance  further  differs  from  the  a-body 
in  containing  much  more  sulphur,  1.27  per  cent.,  as  compared  with 
0.56  per  cent.,  which  is,  moreover,  in  part  at  least,  present  in  a 
loosely  combined  form,  while  the  entire  quantity  that  is  found  in  the 
a-crystalline  is  firmly  combined. 

That  these  bodies  are  intimately  concerned  in  the  concentration  of 
the  light  cannot  be  doubted.  The  refractive  index  of  the  inner 
layer  of  the  lens,  in  man,  is  given  as  1.407,  while  that  of  the 
central  portion  is  1.456. 

Of  the  significance  of  the  fats,  lecithins  and  cholesterins  in  the 
lens,  nothing  is  known,  but  it  appears  that  the  amount  of  the  two 
latter,  at  least,  is  much  increased  in  senile  cataract,  while  the 
quantity  of  the  albumins,  as  a  whole,  is  diminished.  The  albumoid, 
however,  is  then  possibly  increased. 

The  Vitreous  Body". — The  vitreous  body  of  the  eye  is  a  jelly- 
like material,  which  consists  of  a  fine  framework  of  collagen,  enclos- 
ing the  liquid  portion  of  the  body  proper.  This  presents  an  alka- 
line reaction,  and  contains  only  a  very  small  amount  of  solids.  Its 
general  composition  is  seen  below  : 

Pro  mille. 

Water 989.00 

Solids 11.00 

Albumin 0.70 

Urea 0.64 

Paralactic  acid      traces. 

Glucose      traces. 

Mineral  salts 9.00 

Among  the  albumins  present  Morner  claims  to  have  found  a 
hyalomucoid,  which  is  closely  related  to  the  corneal  mucoid,  but 
contains  12.27  per  cent,  of  nitrogen  and  1.19  per  cent,  of  sulphur, 
as  compared  with  12.79  per  cent,  of  nitrogen  and  2.07  per  cent,  of 
sulphur  in  the  case  of  the  latter. 

The  Retina. — A  general  idea  of  the  chemical  composition  of  the 
retina  may  be  formed  from  the  following  analyses,  which  are  taken 
from  Cahn  : 


THE  EYE.  409 

Horse.  Ox. 

Water 89.99  86.52-87.61 

Solids 10.01  13.43-12.39 

Soluble  albumins 4.35  "I  q  .r     f- ^o 

Insoluble  albumins 1.36  J  ^•*^"  ^'""^ 

Extractives 0.67  0.67-  1.07 

Cholesterin  \       0.65-  0.77 

Lecithin       \       2.39  2.08-  2.89 

Fats              )       0.00-  0.47 

Soluble  salts 1.11  0.67-  0.93 

Insoluble  salts 0.01  0.02-  0.27 

Like  the  gray  matter  of  the  brain,  from  which  the  retina  is  essen- 
tially derived,  the  membrane  presents  an  acid  reaction  when  per- 
fectly fresh,  but  becomes  alkaline  soon  after  death. 

The  albumins  which  are  found  in  the  retina  appear  to  be  identical 
with  those  of  the  brain  substance,  and  here,  as  there,  we  also  meet 
with  neurokeratin.  This  apparently  forms  the  sheath  of  the  outer 
portion  of  the  rods.  In  their  interior  we  meet  with  protagon, 
lecith-albumins,  and  in  many  animals  with  a  peculiar  red  pigment, 
which  has  been  termed  rhodopsin. 

Rhodopsin. — Of  the  significance  of  this  pigment  nothing  is  known. 
It  occurs  in  the  outer  portion  of  the  rods  and  is  absent  in  the 
cones.  As  the  macula  lutea,  viz.,  the  point  of  clearest  vision,  is 
composed  only  of  cones,  we  may  conclude  that  its  presence  is  not 
essential  to  sight,  and,  as  I  have  just  said,  the  pigment  is  not  found 
in  all  animals.  It  is  absent  in  chickens,  pigeons,  in  certain  reptiles, 
bats,  etc.,  but  is  present  in  owls  and  deep-sea  fishes.  On  exposure 
to  daylight  the  pigment  fades,  and,  to  isolate  the  substance,  it  is 
necessary  to  work  with  sodium  light.  If  the  living  retina,  after 
having  been  kept  in  the  dark  for  some  time,  is  suddenly  exposed  to 
an  intense  light,  which  is  broken  in  part  through  the  interposition 
of  some  dark  object,  such  as  the  framework  of  a  window,  and  if  the 
remaining  pigment  is  then  fixed  with  a  4  per  cent,  solution  of  alum, 
red  pictures  of  the  interposed  object  can  be  obtained  on  the  retina, 
while  the  remaining  portion  has  become  decolorized.  Such  pictures 
are  termed  optograms. 

The  regeneration  of  the  pigment,  which  is  constantly  going  on  in 
the  living  animal,  is  apparently  dependent  upon  the  integral  union 
of  the  layer  of  rods  and  cones  with  the  pigmented  epithelial  layer 
of  the  retina ;  but  of  the  manner  in  which  this  restitution  takes 
place,  we  know  nothing.  It  appears,  however,  that  its  formation  is 
preceded  by  the  development  of  a  yellow  pigment,  which  is  termed 
xcoithopsin. 

Of  the  chemical  nature  of  rhodopsin  nothing  is  known.  Be- 
sides daylight,  it  is  decomposed  by  acids,  alcohol,  ether,  chloroform, 
and  solutions  of  the  alkaline  hydrates,  by  heating  to  a  temperature 
of  from  52°  to  53°  C.  for  several  hours,  or  instantaneously  at  76°  C. 
Toward  ammonia  and  a  solution  of  alum  it  is  refractory. 

It  is  easily  soluble  in  water,  containing  from  2  to  5  per  cent,  of 
Platner's  bile.     From  such  a  solution  it  is  precipitated  on  dialysis 


410  THE  EYE  AND   THE  EAR. 

or  by  salting  with  ammonium  sulphate  or  magnesium  sulphate.  The 
substance  then  appears  as  a  violet  amorphous  material.  On  spec- 
troscopic examination  no  specific  bands  are  observed,  but  merely  a 
general  absorption  between  D  and  C,  which  is  especially  marked 
about  E. 

Cliromophanes. — Chromophanes  are  pigments  which  apparently 
belong  to  the  lipochromes,  and  are  found  in  the  retinal  cones  of 
reptiles  and  birds.  They  here  occur  in  the  form  of  red,  green,  and 
yellow  oil  globules,  which  are  quite  distinct,  and  are  situated  at  the 
inner  ends  of  the  cones.  The  pigments  in  question  are  termed  rhodo- 
phane,  chlorophane,  and  xanthophane,  respectively.  Unlike  the 
pigment  of  the  rods,  these  chromophanes  are  apparently  not  affected 
by  light,  unless  exposed  for  several  days.  Of  their  significance, 
nothing  is  known. 

The  epithelial  layer  of  the  retina,  wliich  adjoins  the  choroid,  con- 
tains a  black  pigment,  which  is  probably  identical  with  that  of  the 
choroid.  This  is  ievvaeA  fuscin,  and  belongs  to  the  class  of  the  mel- 
anins,  which  comprise  the  black  pigments  that  are  found  in  the  hair, 
in  the  negro-skin,  in  melanotic  tumors,  etc.  According  to  Lan- 
dolt,  this  particular  form  contains  C,  54.48  per  cent.  ;  H,  5.36 ; 
N,  12.65;  O,  27.52.  In  addition  iron  is  also  found,  and  the 
opinion  has  been  expressed  that  the  substance  may  be  derived  from 
the  coloring-matter  of  the  blood.  This  supposition  is  strengthened 
by  the  observation  that  the  first  appearance  of  the  pigment  in  the 
embryo  coincides  in  point  of  time  with  the  development  of  the 
choroidal  bloodvessels. 

Besides  fuscin,  a  pigment  of  a  yellow  color  has  also  been  found 
in  the  pigmented  epithelial  lining  of  the  retina,  which  is  termed 
Upochrin.     It  is  apparently  a  yellow  lipochrome. 

The  Choroid. — Aside  from  the  common  components  of  connec- 
tive tissue,  the  choroid,  as  has  just  been  mentioned,  contains  a  black 
pigment,  fuscin,  which  is  probably  identical  with  that  found  in  the 
pigmented  epithelial  lining  of  the  retina  (see  above). 

THE   EAR. 

The  chemical  composition  of  the  organic  portion  of  the  middle 
and  the  internal  ear  has  not  as  yet  been  studied.  The  perilymph 
and  endolymph  present  an  alkaline  reaction,  and,  in  addition  to  the 
common  mineral  salts  of  the  lymph,  contain  traces  of  albumin,  and 
in  some  animals  a  mucinous  body  of  unknown  character. 


CHAPTEE    XIX. 

THE  SUPPORTING  TISSUES. 

In  contradistinction  to  those  tissues  of  the  animal  body  which  are 
essentially  composed  of  cells,  and  in  which  the  albumins  proper  con- 
stitute the  greater  portion  of  the  organic  solids,  we  find  that  in  the 
so-called  supporting  tissues  which  comprise  the  common  connective 
tissues,  cartilage,  and  bone,  the  albuminoids  stand  in  the  foreground. 
Their  preponderance  here  coincides  with  the  extensive  development 
of  the  matrix,  while  the  cellular  elements  enter  into  the  histological 
picture  to  a  more  or  less  insignificant  extent.  This  statement,  how- 
ever, holds  good  only  for  the  higher  animals,  and  more  specifically 
for  the  fully  developed  animals.  In  lower  forms  of  life,  and  during 
the  embiyonic  stage  of  the  development  of  the  higher  forms,  these 
structures  are  rich  in  cells,  and  we  find  then  an  underlying  matrix 
in  which  a  diiferentiation  into  supporting  tissue  proper  has  not  as 
yet  occurred  or  exists  to  only  a  limited  extent.  Such  tissue  is 
termed  embryonic  connective  tissue,  and  is  also  known  as  inucous 
tissue.  In  the  adult  animal  it  is  found  only  in  the  vitreous  humor 
of  the  eye.  In  typical  form  it  is  seen  in  the  umbilical  cord,  in 
which  it  constitutes  the  so-called  jelly  of  AVharton.  The  matrix  is 
here  very  rich  in  water,  and  contains  a  mucinous  substance,  which 
is  soluble  in  a  0.5  pro  mille  solution  of  hydrochloric  acid.  In  addi- 
tion, traces  of  albumin  are  met  with,  while  collagen  is  usually  absent. 
Of  the  composition  of  the  cells  nothing  specific  is  known,  but  it  is 
quite  likely  that  their  processes  consist  of  collagen. 

White  Fibrous  Tissue. — The  fibrils  of  white  fibrous  tissue  con- 
sist of  collagen,  and  are  bound  together  by  a  cement-substance, 
which  represents  the  original  undifferentiated  matrix.  As  in  the 
case  of  the  embryonic  connective  tissue,  this  contains  traces  of  the 
common  albumins  of  the  plasma,  and  a  mucinous  substance,  Avhich, 
in  contradistinction  to  that  of  the  umbilical  cord,  is  insoluble  in 
a  0.5  pro  mille  solution  of  hydrochloric  acid.  Analysis  of  this 
substance  has  given  the  following  results  :  C,  48.30;  H,  6.44;  N, 
11.75  ;  S,  0.81  ;  and  O,  32.70  per  cent.  According  to  Lobisch,  its 
formula  is  CiguHj^e^gjSO^p.  To  isolate  the  body  in  question,  liga- 
ments, such  as  the  tendo  Achillis,  are  cut  into  small  pieces  and 
first  extracted  with  cold  water,  which  dissolves  the  albumins  and 
a  small  fraction  of  the  mucin.  The  remaining  material  is  then 
placed  in  a  half-saturated  solution  of  lime-water,  in  which  the 
mucin  is  readily  soluble.  After  filtering,  the  substance  is  precipitated 
by  adding  an  excess  of  acetic  acid  (see  page  124).    The  residual  sub- 

411 


412  THE  SUPPORTING   TISSUES. 

stance,  after  removal  of  the  mucin,  consists  of  tlie  collagen-fibrils, 
a  few  cellular  elements,  and  quite  commonly  also  contains  isolated 
fibrils  of  the  yellow  or  elastic  variety,  which  can  be  readily  recog- 
nized on  microscopical  examination  by  tlieir  higher  power  of  refrac- 
tion. When  placed  in  water,  or,  still  better,  in  a  dilate  solution  of 
acetic  acid  or  caustic  alkali,  the  white  fibres  swell,  while  solutions 
of  some  of  the  metallic  salts,  such  as  ferric  sulphate  and  mercuric 
chloride,  cause  them  to  shrink.  Tannic  acid  acts  in  a  similar 
manner.  Owing  to  the  great  stability  of  the  compound  of  the  latter 
with  collagen,  tannic  acid  is  extensively  utilized  in  the  preparation 
of  leather. 

On  boiling  white  fibrous  tissue  in  water  the  collagen  dissolves, 
with  the  formation  of  gelatin,  which  latter  separates  as  a  jelly-like 
mass  on  cooling. 

Yellow  or  Elastic  Tissue. — In  the  yellow  ehistic  fibres,  elastin 
takes  the  place  of  the  collagen  of  the  white  fibrous  variety.  For 
purposes  of  study,  the  substance  is  most  conveniently  obtained  from 
the  ligamentum  nuchse  of  the  ox,  in  which  such  fibres  are  almost 
exclusively  found. 

Reticulated  Tissue. — In  the  reticulated  tissue,  which  constitutes 
the  fibrous  framework  of  the  lymph-glands  of  the  body,  but  which 
is  also  found  in  the  alveoli  of  the  lungs,  in  the  liver,  the  kidneys, 
and  the  intestinal  mucous  membrane,  the  fibres  consist  of  reticulin. 

Reticulin  is  said  to  have  the  composition  C,  52.88  ;  H,  6.97 ;  N, 
15.63;  S,  1.88;  P,  0.34.  It  is  insoluble  in  water,  alcohol,  ether, 
dilute  mineral  acids,  lime-water,  and  solutions  of  sodium  carbonate. 
It  resists  the  action  of  pepsin  and  trypsin,  and  is  dissolved  only  in 
cold  sodium  hydrate  solution  on  standing  for  several  weeks.  It 
does  not  give  Millon's  reaction,  and  accordingly  yields  no  tyrosin  on 
hydrolytic  decomposition.  On  prolonged  boiling  with  water  or 
dilute  alkalies,  its  phosphorus  is  split  off;  the  residual  material  is 
then  soluble  in  water,  and  can  be  precipitated  from  its  solutions  by 
means  of  acetic  acid. 

CARTILAGE. 

Histologically  considered,  cartilage  consists  of  a  more  or  less 
hyalin  matrix,  in  Avhich  a  variable  number  of  cartilage-cells  are  found 
imbedded  In  certain  localities,  further,  a  differentiation  of  the  mat- 
rix into  fibres,  both  of  the  white  and  the  yellow  elastic  variety,  is 
observed.  Such  fibres,  as  in  the  case  of  the  corresponding  connec- 
tive tissue,  consist  of  collagen  and  elastin,  respectively. 

Of  the  composition  of  the  cells  nothing  specific  is  known.  Appar- 
ently they  contain  a  small  amount  of  glycogen,  which  disapjK'ars 
during  starvation.  Traces  of  fat  are  also  found.  During  embryonic 
life  they  are  quite  numerous,  but  later  they  diminish  in  number,  and 
in  the  adult  animal  the  matrix  largely  predominates.  Embryonic 
cartilage  does  not  yield  gelatin  on  boiling  with  water,  and  it  is  quite 
likely  that  as  in  the  case  of  the  matrix  of  embryonic  connective 


CARTILAGE.  413 

tissue  the  matrix  here  also  consists  essentially  of  water  and  some 
mucinous  substance.  Whether  or  not  this  is  identical  with  the  so- 
called  chondromucoid,  which  can  be  obtained  from  the  cartilage  of 
the  adult  animal,  is  not  known. 

A  general  idea  of  the  chemical  composition  of  cartilage  may'  be 
formed  from  the  following  analyses,  wdiich  are  taken  from  His : 

Costal  cartilage  Articular  cartilage 

(human).  from  knee-joint  (human). 

Water 67.67  per  cent.  73.59  per  cent. 

Solids 32.33   "      "  26.41    "      " 

Organic  material    ....  30.13   "       "  24.87    "       " 

Mineral  salts 2.20   "       "  1.54   "       " 

Analysis  of  the  mineral  salts  has  given  the  following  results 
(calculated  for  100  parts  of  the  mineral  ash) : 

Sodium  chloride 6.11    per  cent.  22.48  per  cent. 

Sodium  sulphate 44.81      "       "  55.17   "       " 

Potassium  sulphate 26.66      "       " 

Sodium  phosphate      8.42      "       "  7.39   "       " 

Calcium  phosphate 7.88  "I    <(       «  j-  ^-j^    «       « 

Magnesium  phosphate       .    .    .  4.55  J 

The  organic  constituents  of  the  cartilaginous  matrix  are  essentially 
represented  by  chondroitin-sulphuric  acid  as  such,  and  its  compounds 
with  collagen  and  albumins.  In  addition,  a  small  amount  of  soluble 
albumins  is  found,  as  also  a  peculiar  insoluble  albuminous  sub- 
stance, which  has  been  termed  albumoid.  Handel  further  found 
0.2168  per  cent,  of  glycogen,  viz.,  much  larger  amounts  than  are 
obtained  from  any  of  the  other  skeletal  parts. 

Chondroitin-sulphuric  Acid. — This  substance  is  a  conjugate 
sulphate,  and,  according  to  Schraiedeberg,  has  the  composition 
CigHoyNOi^.SOg.  On  hydrolytic  decomposition  it  yields  a  hyalin, 
chondroitin,  which  in  turn  gives  rise  to  the  formation  of  chondrosin. 
The  chondrosin,  according  to  Schmiedeberg,  can  further  give  rise 
to  glucuronic  acid  and  glucosamin.  But  this  has  been  disproved 
(Neubauer,  Orgler-Neuberg).  Neuberg  obtained  a  substance  on 
hydrolysis  of  chondrosin,  which  was  shown  to  be  tetra-oxyamido- 
capron'ic  acid  [C6H702(OH)4(NH2)].  This  is  in  turn  is  combined 
in  the  chondrosin  with  a  carbohydrate-like  substance  of  unknown 
composition. 

(1)  C,«H„N0„.S03   +   HP  =  C,8H,,N0h   +  H^SO, 

Chondroitin-sulphuric  Chondroitin. 

acid. 

(2)  CigH^NOu   +   3B.fi        =  C,2H,,N0„    +  3CH3.COOH 
Chondroitin.  Chondrosin.  Acetic  acid. 

(3)  C,2H.,iN0u  +   H^O  =   C6HA(OH),(NH2)+i 

Chondrosin. 

Both  chondroitin  and  chondrosin  are  monobasic  acids.  The  latter 
reduces  Fehling's  solution  directly,  while  in  the  case  of  chondroitin 
this  occurs  only  after  the  substance  has  been  decomposed. 

Chondroitin-sulphuric    acid   is    an  amorphous    substance,  and  is 


414  THE  SUPPORTING   TISSUES. 

soluble  in  water.  A  concentrated  solution  resembles  mucilage  in 
appearance  and  consistence.  Its  salts  are  also  for  the  most  part 
soluble  in  water.  The  sodium  and  potassium  salts  can  be  pre- 
cipitated by  means  of  ferric  chloride,  lead  subacetate,  and  alcohol, 
while  silver  nitrate,  zinc  chloride,  tannic  acid,  and  potassium  ferro- 
cyanide,  the  latter  in  the  presence  of  acetic  acid,  are  without  effect. 

In  the  cartilage  its  potassium  and  sodium  salts  occur  both  as  such 
and  in  combination  with  collagen  and  albumins.  A  mixture  of  these 
compounds,  according  to  Schmiedeberg,  constitutes  the  so-called 
chondromucoid  of  Morner.  If  a  solution  of  gelatin  is  mixed  with 
an  acidified  solution  of  the  potassium  or  sodium  salts  of  the  acid, 
a  precipitate  occurs.  This  also  results  if  cartilage  is  boiled  with 
water  and  the  resulting  impure  solution  of  gelatin,  which  was 
formerly  termed  chondrin,  is  acidified  with  a  dilute  mineral  acid. 
The  precipitate  consists  of  the  free  chondroitin-sulphuric  acid,  and 
is  soluble  in  an  excess  of  the  acid. 

Chondroitin-sulphuric  acid,  while  essentially  a  constituent  of 
hyaline  cartilage,  has  also  been  found  in  elastic  and  fibrous  carti- 
lage, in  bone,  in  the  inner  coats  of  the  larger  arteries,  in  the  chon- 
dromas, in  the  amyloid  substance  obtained  from  liver,  in  the 
mucosa  of  the  pig's  stomach,  and  in  the  ligamentum  nuchte.  Traces 
are  likewise  found  in  the  urine. 

Isolation. — To  isolate  chondroitin-sulphuric  acid  from  cartilage 
shavings,  the  material  is  boiled  with  a  5  per  cent,  solution  of  caustic 
alkali.  The  solution  is  neutralized,  and  freed  by  filtration  from 
the  alkaline  albuminates  which  have  been  formed  during  the  process 
of  boiling.  Albumoses  are  removed  by  means  of  tannic  acid,  the 
excess  of  the  latter  by  means  of  lead  subacetate,  and  the  excess 
of  lead  with  hydrogen  sulphide.  The  acid  is  then  precipitated  with 
alcohol.  To  purify  the  substance,  it  is  dissolved  in  water,  and  the 
solution  dialyzed  and  reprecipitated  with  alcohol.  The  solution  ia 
water  and  precipitation  with  alcohol  is  repeated  several  times,  when 
the  acid  is  finally  washed  with  alcoholic  ether. 

Isolation  of  Chondromucoid. — To  isolate  the  chondromucoid, 
viz.,  the  compounds  of  chondroitin-sulphuric  acid  with  collagen 
and  albumins,  the  cartilage  shavings  are  first  extracted  with  w^ater, 
which  dissolves  the  free  chondroitin-sulphuric  acid  and  a  small 
amount  of  the  chondromucoid.  On  acidulating  this  solution  with 
a  3  pro  raille  solution  of  hydrochloric  acid  and  heating  on  a  water- 
bath,  the  chondromucoid  is  gradually  precipitated,  while  the  free 
acid  remains  in  solution.  The  cartilaginous  residue  is  then  ex- 
tracted with  a  2  to  3  pro  mille  solution  of  hydrochloric  acid  at  a 
temperature  of  35°  to  40°  C,  which  dissolves  any  collagen  that 
may  be  present  as  such.  After  washing  with  water  the  remaining 
material  is  extracted  with  a  5  pro  mille  solution  of  caustic  alkali. 
The  chondromucoid  is  thus  dissolved,  and  is  then  precipitated  with 
an  acid.  After  repeated  solution  in  an  alkali  and  precipitation 
with  an  acid  it  is  finally  washed  with  alcohol  and  ether. 


BONE.  415 

Albumoid. — The  albumoid  which  is  found  in  the  cartilage  of 
adult  animals  is  apparently  closely  related  to  elastin  and  keratin,  but 
differs  from  the  latter  in  containing  sulphur,  and  from  the  former  in 
its  digestibility  by  gastric  juice.  It  gives  the  common  color-reac- 
tions of  the  albumins,  but  is  insoluble  in  all  neutral  solvents,  and 
dissolves  in  acids  and  alkalies  only  with  great  difficulty. 

Isolation. — To  isolate  the  substance,  cartilage  shavings  are  first 
extracted  with  a  0.5  per  cent,  solution  of  caustic  alkali,  to  remove 
the  chondroraucoid,  and  the  free  chondroitin-sulphuric  acid.  The 
remaining  material  is  then  washed  with  water  and  boiled  with 
water  in  a  Papin  digester.  Any  collagen  that  may  be  present  is 
thus  dissolved,  while  the  albuminoid  together  with  the  cartilage-cells 
remains  behind. 

Mineral  Constituents. — Among  the  mineral  constituents  of  car- 
tilage, the  very  large  amount  of  alkaline  sulphates  is  especially 
noteworthy.  These  are  supposedly  not  present  in  the  free  state, 
however,  beyond  traces  perhaps,  but  result  from  the  chondroitin- 
sulphate  on  incineration.  In  the  cartilage  of  the  shark,  very  curi- 
ously, sodium  chloride  constitutes  as  much  as  94.2  per  cent,  of  the 
total  amount  of  mineral  ash.  As  this  represents  17.7  per  cent,  of 
the  moist  material,  the  amount  of  sodium  chloride  would  be  suffi- 
cient to  form  a  concentrated  solution  in  the  cartilage,  which,  of 
course,  is  scarcely  conceivable  as  occurring  in  living  tissue.  It  is 
hence  assumed  that  the  salt  is  present  in  organic  combination,  but 
of  its  pairling  nothing  definite  is  known.  According  to  Bunge,  such 
large  amounts  of  sodium  chloride  are  also  found  in  mammals  during 
the  period  of  intra-uterine  life  and  shortly  after  birth,  and  he  be- 
lieves that  this  is  in  accordance  with  the  biogenetic  law  which  under- 
lies the  development  of  the  higher  forms  of  life  from  those  of  a 
lower  order. 

With  the  appearance  of  old  age  a  gradual  deposition  of  calcium 
salts  occurs  in  the  matrix  of  the  cartilage,  so  that  partial  ossifica- 
tion takes  place.  This,  of  course,  also  occurs  during  the  develop- 
ment of  normal  bone,  but  it  is  to  be  noted  that,  in  contradistinction 
to  true  bone,  the  matrix  of  senile,  ossified  cartilage  retains  its 
original  characteristics, 

BONE. 

The  matrix  of  bone-tissue,  like  its  contained  fibrils,  is  com- 
posed of  collagen,  which  is  here  termed  ossein,  and  is  supposedly 
identical  with  the  common  form  that  is  obtained  from  connective 
tissue.  Of  the  composition  of  the  cells,  viz.,  the  so-called  bone- 
corpuscles,  nothing  is  known.  With  their  processes  they  occupy  the 
lacunae  and  canaliculi,  and  are  separated  from  the  bony  structure 
proper  by  a  layer  of  a  very  resistant  albuminous  substance  of 
unknown  character. 

In  contradistinction  to  the  other  supporting  tissues  of  the  body 


416 


THE  SUPPORTING   TISSUES. 


which  have  thus  far  been  considered,  bone-tissue  apparently  con- 
tains no  ghicoproteids. 

The  function  of  the  bone-tissue,  as  the  principal  supporting  tissue 
of  the  body,  finds  its  expression  in  the  preponderance  of  the  mineral 
constituents  over  the  organic  solids,  and  it  is  interesting  to  note  that 
the  ratio  between  the  two  is  fairly  constant,  not  only  in  different 
bones,  but  also  in  different  animals.  These  salts  are  largely  repre- 
sented by  calcium  phosphate  and  carbonate,  which  impregnate  the 
entire  matrix.  In  addition,  we  find  magnesium  phosphate  and 
small  amounts  of  calcium  chloride,  calcium  fluoride,  potassium  and 
sodium  salts,  and  a  little  iron.  Of  the  manner  in  which  the  differ- 
ent salts  are  combined  with  each  other,  nothing  definite  is  known, 
but  we  may  possibly  assume,  with  Gabriel,  that  the  composition  of 
the  bone-ash,  as  well  as  the  tooth-ash,  can  be  represented  by  the 
formula  [CXCPO,)^  +  CagHPgOig  +  H.O],  in  which  2  to  3  per  cent, 
of  calcium  is  replaced  by  magnesium,  potassium,  and  sodium,  and 
4  to  6  per  cent,  of  the  phosphoric  acid  by  carbonic  acid,  chlorine, 
and  fluorine. 

Whether  or  not  the  mineral  constituents  of  the  bone  exist  in  com- 
bination with  the  organic  components  of  the  tissue  has  not  as  yet 
been  definitely  ascertained,  l)ut  does  not  appear  improbable. 

An  idea  of  the  quantitative  distribution  of  the  different  salts  in 
different  animals  and  bones  may  be  formed  from  the  accompanying 
analyses.  The  figures  have  reference  to  100  parts  of  bone-ash 
(Zalefsky) : 

Human.         Ox.  Turtle,     '^^^f^^' 

Calcium  pho.sphate(Ca3(P04).,)      .    .  83.89  86.09  85.98  87.38 

Magnesiumphosphate(Mg3(Pb4)2)   .  1.04  1.02  1.36  1.05 
Calcium   in  combination  Avith  carbon 

dioxide,  chlorine,  and  fluorine     .    .  7.65  7.36  6.32  7.03 

Carbon  dioxide  1 5.73  6.20  5.27  .    . 

Chlorine 1.80  2.00 

Fluorine^ 2.30  3.00  2.00  .    . 

Calcium         Phosphoric     Magnesium 
oxide  acid  oxide 

(CaO).  (PaOs).  (MgO). 

Small  flounder  (ash  in  general)  .    .  53.13  42.72  0.91 

Man  (ash  in  general) 52.83  38.73  0.48 

Man  (humerus) 51.31  36.65  0.77 

Ox  (femur)      51.28  37.46  1.05 

Goose  (ash  in  general) 51.01  38.19  1.27 

Rabbits,  varying  in  age    between 

one  day  and  four  years  (general 

ash) 51.91-52.89     39.78-42.20    0.83-1.38 

The  variations  in  the  amount  of  bone-ash,  as  a  whole,  in  different 
bones  of  the  same  animal,  are  seen  in  the  following  table  (Fremy) : 

1  These  figures  are  somewhat  too  low,  as  a  certain  amount  of  carbon  dioxide  escapes 
during  the  incineration  of  the  bone. 

2  According  to  Gabriel,  the  amount  of  fluorine  does  not  exceed  0.1  per  cent.,  and  is  usually 
less  than  0.5  per  cent. 


THE  TEETH.  417 

Per  cent. 

Femnr 

Humerus      

Tibia 1-64.1-64.6 

Occipital  bone | 

Cranium J 

Scapula r,3.3 

Vertebrae 54.2 

The  amount  of  water  which  is  found  in  bones  varies  between  13.8 
and  44.3  per  cent.  It  is  greater  in  the  spongy  bones  than  in  those 
of  the  compact  variety,  and  gradually  diminishes  with  age. 

The  bone-marrow  is  pervaded  by  a  network  of  connective  tissue, 
which  is  partly  of  the  white  fibrous  variety  and  partly  reticulated. 
In  its  meshes  we  find  the  cellular  elements  of  the  marrow,  viz., 
the  so-called  myeloplaxes,  the  juvenile  forms  of  the  polynuclear 
neutrophilic  and  eosinophilic  leucocytes,  viz.,  the  myelocytes,  red  cor- 
puscles in  various  stages  of  development,  and  a  variable  number  of 
fat  cells.  These  latter  are  especially  numerous  in  the  so-called  yellow 
vutrroic,  where  the  amount  of  fat  may  represent  as  much  as  96  per 
cent,  of  the  entire  substance.  It  consists  of  olein,  pahuitin,  and 
stearin.  The  red  marrow,  on  the  otlier  hand,  contains  a  much  smaller 
amount  of  fat,  and  owes  its  color  to  large  numbers  of  red  corpuscles. 
It  contains  albumins,  of  which  one  is  regarded  as  a  globulin,  and  is 
said  to  coagulate  at  50°  C.  But  especially  interesting  is  the  pres- 
ence of  peculiar  iron  compounds  which  are  as  yet  but  little  known, 
but  probably  belong  to  the  nucleo-albumins  and  iron-containing 
albuminates.  Their  presence  is  no  doubt  intimately  associated  with 
the  formation  of  red  corpuscles.  Among  the  extractives  of  bone- 
marrow  we  notably  find  lactic  acid  and  hypoxanthin. 

THE   TEETH. 

The  dentin  of  the  teeth  is  a  peculiarly  modified  form  of  bone- 
tissue,  and  is  likewise  composed  of  an  organic  matrix,  which  con- 
sists of  collagen  and  is  impregnated  with  mineral  salts.  The  latter 
are  here  even  more  abundant  than  in  true  bone,  and  represent 
from  64  to  68  per  cent,  of  the  fresh  tissue,  while  of  organic  matter 
we  find  between  26  and  28  per  cent.,  thus  leaving  10  per  cent, 
for  water.  The  relative  distribution  of  the  individual  salts  is  about 
the  same  as  in  common  bone. 

Tlie  dentinal  tubules,  like  the  lacunse  and  canaliculi,  are  appar- 
ently lined  by  the  same  albuminous  substance. 

The  cement  which  surrounds  the  dentin  of  the  root  as  far  as 
the   neck  of  the  tooth   consists  of  true  bone. 

The  enamel,  in  accordance  with  its  epithelial  origin,  contains  no 
collagen.  It  is  very  rich  in  lime  salts,  and  its  mineral  constituents 
represent  as  much  as  96  per  cent,  of  the  total  substance.  Its  organic 
components  correspond  to  about  3.6  per  cent.,  but  are  as  yet 
unknown.     Water  is  practically  absent. 

Other  tissues  which  are  closely  related  to  bone  are  ivory,  tortoise- 
27 


418  THE  SUPPORTING   TISSUES. 

shell,  and  the  scales  of  fishes.  In  the  two  former  the  mineral  con- 
stituents predominate  over  the  organic  matter,  as  in  bone,  while  in 
the  latter  more  organic  material  is  found.  Ivory  is  especially  rich 
in  magnesium  phosphate,  of  which  it  contains  about  15.72  per  cent. 
The  outer  surflice  of  tortoise-shell  is  covered  with  a  layer  of  kera- 
tinized epidermis.  In  fish-scales  mineral  material,  collagen,  and  a 
peculiar  albuminoid  body,  which  is  termed  ichihylepidin,  are  found. 
According  to  M5rner,  this  gives  an  intense  Millon  reaction  and 
contains  a  large  amount  of  loosely  combined  sulphur.  Green  and 
Tower  were  able  to  demonstrate  its  presence  in  a  large  numl^er  of 
different  fishes.  It  is  apparently  found  in  most  of  the  teleosts,  but 
is  absent  in  the  elasmobranchs. 

In  the  invertebrate  animals,  with  the  exception  of  the  cephalopods, 
and  possibly  also  the  branchiopods,  collagen  is  not  found  The 
internal  supporting  structures  are  here  represented  by  ingrowths  of 
the  cuticular  formations,  which  are  derived  from  the  epidermal  cells, 
and  consist  largely  of  skeletins  and  It//aIo(/ens  which  have  become 
impregnated  with  lime  salts.  Closely  related  to  the  latter  is  chitin, 
which  enters  largely  into  the  composition  of  the  outer  skeleton 
of  the  arthropods  and  their  nerve-sheaths.  Associated  with  it  we 
find  the  so-called  tunicin,  which  is  regarded  as  a  cellulose,  and  which 
is  found  in  especial  abundance  in  the  mantle  of  the  tunicates. 

ADIPOSE    TISSUE. 

Adipose  tissue  may  be  regarded  as  a  special  form  of  connective 
tissue  in  which  the  cellular  elements  enclose  globules  of  fat.  AVhen 
fully  developed,  the  individual  cells  appear  as  greatly  distended 
vesicles,  which  are  covered  by  a  cell-membrane.  The  original  proto- 
plasm has  been  almost  entirely  replaced  by  fat,  and  occurs  merely 
as  a  thin  layer  beneath  the  membrane.  The  nucleus  also  has 
been  displaced  to  the  periphery,  and  can  scarcely  be  discerned  with- 
out special  methods  of  staining.  Such,  fat-cells  usually  occur  in 
groups,  and  are  held  together  by  delicate  fibres  of  connective  tissue, 
in  the  meshes  of  which  a  network  of  blood-capillaries  is  found 
surrounding  each  cell.  When  large  numbers  of  fat-cells  occur, 
the  individual  groups  are  gathered  into  lobules,  and  these  into 
lobes.  In  the  living  tissue  the  contained  fat  exists  in  a  liquid 
form,  but  congeals  after  death,  and  is  then  more  or  less  solid  accord- 
ing to  the  character  of  the  individual  fats.  Stearin  and  palmitin 
then  often  separate  out  in  crystalline  form.  Of  the  chemical  com- 
position of  the  original  cells,  before  their  invasion  with  fat-glol^ules, 
nothing  definite  is  known.  They  contain  albumin  and  are  appar- 
ently rich  in  water.  The  cell-membrane  is  exceedingly  resistant  to 
solvents,  but  is  digested  by  the  gastric  juice,  and  possibly  consists 
of  an  elastin-like  substance. 

The  relative  amount  of  water  and  fat  Mhich  is  found  in  adipose 
tissue  varies  primarily  with  the  state  of  nutrition,  and  differs  in 
different  animals. 


ADIPOSE  TISSUE.  419 

The  fats  in  question  are  principally  the  triglycerides  of  stearic 
acid,  palmitic  acid,  and  oleic  acid.  Others,  such  as  the  glycerides 
of  capronic  acid  and  valerianic  acid,  are  not  constant  constituents  of 
adipose  tissue,  but  are  met  with  only  exceptionally,  and  always  in 
very  small  amounts.  In  man,  a  comparatively  large  amount  of 
oloin  is  found,  but  it  is  not  so  abundant  as  in  certain  cold-blooded 
animals,  in  which  it  may  form  the  greater  portion  of  the  fat.  The 
quantitative  relation  between  the  tliree  forms  is  by  no  means  con- 
stant in  all  parts  of  the  body,  so  that  the  melting-point  of  the  fats 
from  different  regions  may  be  quite  different.  It  differs,  moreover, 
in  different  animals.  This  is  shown  in  the  following  table,  which 
is  taken  from  Gautier  : 

Mutton  (subcutaneous)       27°-31°  C. 

Mutton  (perirenal)      37°-43°  C. 

Mutton  (epiploic)        36°-39°  C. 

Man  (panniculus  adiposus) 15°-22°  C. 

Man  (perirenal ) 25°  C. 

Dog 20°-22.5° 

Ox       39°  C. 

Bone-marrow  of  ox 45°  C. 

Calf 52°  +  C. 

Horse 31°  +  C. 

Pig 40°  C. 

Duck      35°  C. 

Of  special  interest  is  the  fact  that  it  is  possible  to  replace  the  com- 
mon fats  of  one  animal  by  those  of  another,  and  even  by  fats  which 
are  not  foimd  normally  in  the  animal  world.  If  dogs,  in  ^\'hich  the  fats 
have  been  removed  by  starvation,  are  thus  fed  with  vegetable  fats,  such 
as  rape-oil,  this  is  subsequently  found  in  the  tissues  of  the  animal, 
and  may  be  recognized  by  its  low  melting-point  (23°  C)  and  the 
presence  of  the  glyceride  of  erucic  acid.  In  a  similar  manner  a 
deposition  of  mutton  tallow  may  be  effected,  which  begins  to  melt 
at  about  40°  C,  while  the  common  fat  of  dogs  melts  at  20°  C. 

In  addition  to  the  fats,  small  amounts  of  lecithin,  cholesterin,  and 
free  fatty  acids  may  also  be  isolated  from  adipose  tissue.  We 
further  find  a  yellow  lipochrome,  to  which  the  color  of  the  fat  is 
due.  General  analysis  of  human  fat  has  given  the  following  results 
(Jaeckli) : 

Fat  from  subcutaneous      Fat  from  child         f.„t  -.„_^  ij^o,^^,, 
tissue.  three  days  old.         ^  ^^  ^'^^^  lipoma. 

Olein      72.1-85.3  per  cent.      55.0  per  cent.     68. 6-90. 2  per  cent. 

Corresponding  amount  of 

oleic  acid  ......  69.6-S1.6       "  52.7  "             65.7-86.4 

Palmitic  acid 16.9-21.1        "  .    .  7.8-24.9       " 

Stearic  acid 4.9-6.3         "  .    .  1.5-5.9 

Free  acid  (calculated  as 

oleic  acid) 0.19-0.52      "  0.36  "              0.15-0.34     " 

Phosphoric  acid  (PA)      0.006-0.007"  .    .  0.001-0.63   " 

Lecithin    ......".    0.07-0.08     "  .    .  0.01-7.21      " 

Pure  cholesterin  ....    0.24               "  .    . 

Ivon-saponifiable  material  [  0.34-1.69 

exclusive  of  cholesterin  0.08               "  .    . 


420  THE  SUPPORTING   TISSUES. 

As  comjxired  with  that  of  otlier  liigher  mammals  human  fat 
shows  no  special  points  of  difference  in  its  composition. 

Analysis  of  Adipose  Tissue. — The  material  in  question  is  first 
dried,  ground  with  a  little  sand,  and  extracted  successively  with 
ether  and  alcohol.  The  alcoholic  extract  is  evaporated  to  dry- 
ness, the  residue  washed  with  water  to  remove  the  soluble  salts,  and 
is  then  extracted  with  ether.  The  ethereal  extracts  are  united,  and 
the  ether  is  distilled  off,  when  the  fats,  lecithins,  cholesterins,  free  flitty 
acids,  and  the  lipochromes  remain.  The  fatty  acids  are  transformed 
into  their  salts  by  adding  a  slight  excess  of  sodium  carbonate,  and 
heating  to  a  temperature  of  100°  C.  The  resulting  soaps  are  ex- 
tracted with  water.  The  insoluble  portion  is  dissolved  in  ether,  the 
ether  is  distilled  off,  and  the  residue  is  heated  on  a  water-bath  with 
an  alcoholic  solution  of  sodium  hydrate,  which  saponifies  the  fats. 
The  resulting  material  is  extracted  with  ether,  which  dissolves  the 
cholesterin.  The  insoluble  residue  is  dissolved  in  water,  and  the 
solution  is  saturated  with  carbon  dioxide  and  extracted  with  strong 
alcohol.  This  takes  up  the  soaps  and  the  glycerin.  The  alcoholic 
solution  is  transformed  into  an  aqueous  solution,  in  which  the  soaps 
are  decomposed  with  a  dilute  acid.  The  free  fatty  acids  are  thus 
precipitated,  and  can  then  be  separated  from  each  other  according^ 
to  the  usual  methods. 

Aside  from  adipose  tissue,  fats  are  met  with  in  all  the  organs  of 
the  body,  but,  with  the  exception  of  the  manmiary  glands  during 
their  functional  activity,  they  are  found  normally  only  in  traces. 
Under  pathological  conditions,  however,  notable  quantities  of  fat 
may  be  met  with.  We  then  speak  of  a  fatty  degeneration  of  the 
organs.  This  is  especially  observed  in  the  liver  in  cases  of  acute 
vellow  atrophy,  and  can  also  be  brought  about  artificially  by  poison- 
ing with  phosphorus,  antimony,  arsenic,  etc. 

Among  the  fluids  of  the  body,  large  quantities  are  normally 
found  only  in  the  milk,  and  in  the  chyle  during  the  process  of 
digestion. 

Origin  of  the  Fats. — That  a  portion  of  the  fat  that  is  found 
in  the  animal  body  is  directly  referable  to  the  fats  which  have  been 
ingested  as  such  cannot  be  doubted.  This  is  proved  not  only  by 
the  observation  that  it  is  possible  to  replace  the  fats  which  are 
peculiar  to  a  certain  animal  by  those  of  another,  or  even  by  vegetable 
flits,  as  has  been  shown  above,  but  also  by  the  fact  that  a  gradual 
deposition  of  fats  occurs  in  dogs  which  have  been  starved  and 
are  then  fed  on  very  little  albuminous  material,  but  with  much  fat. 
In  such  cases  it  can  easily  be  proved  that  the  amount  of  albu- 
mins ingested  is  far  too  small  to  be  the  source  of  the  fat  that  has 
been  stored. 

The  ingested  fats,  however,  are  not  the  only  source  of  the  fats 
found  in  the  tissues,  and  there  is  evidence  to  show  that  they  may 
also  be  derived  from  the  albumins  and  the  carbohydrates.     Their 


ADIPOSE  TISSUE.  421 

origin  from  the  former  is  suggested  by  many  observations.  It 
is  thus  well  known  that  the  albuminous  constituents  of  human 
bodies  when  buried  in  moist  ground,  and  notably  the  muscle-tissue, 
may  undergo  a  peculiar  transformation,  which  is  characterized  by 
the  disappearance  of  the  albumins  and  their  replacement  by  free 
fatty  acids  and  the  calcium  and  ammonium  soaps  of  palmitic  acid 
and  stearic  acid,  which  constitute  the  so-called  adipocere,  or  Leichen, 
wax  of  the  Germans.  This  transformation,  however,  is  probably 
brought  about  through  the  activity  of  micro-organisms,  and  does  not 
prove  in  itself  that  in  the  living  animal  an  actual  formation  of 
neutral  fats  can  occur  from  the  albumins.  But  it  shows,  at  all 
events,  that  forces  which  are  at  work  in  tlie  living  world  can  bring 
about  the  formation  of  two  of  the  higher  fatty  acids  at  least  which 
enter  into  the  composition  of  the  common  fat  from  albuminous 
material.  In  the  laboratory  such  a  transformation  has  not  as  yet 
been  accomplished,  if  we  disregard  the  observations  of  E.  Voit,  who 
claims  to  have  noted  the  appearance  of  higher  fatty  acids,  when  43 
grammes  of  albumin  were  kept  in   milk  of  lime  for  twelve  months. 

Proof  of  the  possible  origin  of  fats  from  albumins,  on  the  other 
hand,  seems  to  be  afforded  by  the  phenomena  of  fatty  degenera- 
tion, where  an  actual  deposition  of  large  amounts  of  fat  can  be 
demonstrated  in  the  cells  of  organs  in  which  only  traces  are  nor- 
mally found.  It  has  been  urged,  however,  that  the  fat  which  is  here 
encountered  has  not  developed  in  situ,  but  has  been  carried  to  the 
organs  in  question  from  the  adipose  tissue  proper.  That  such  a 
transposition  of  fats  may  occur  is  possible,  and  has  indeed  been 
proved  for  the  fatty  degeneration  of  the  liver  which  results  from 
poisoning  with  phloridzin  ;  Leick  and  Winckler  could  also  demon- 
strate that  in  doffs  which  had  first  been  starved  and  then  fed  with 
mutton  tallow,  and  finally  poisoned  M'ith  phosphorus,  the  fatty 
degeneration  of  the  heart  muscle  also  was  referable  to  transported 
fat.  This  impression  regarding  the  origin  of  fat  in  fatty  de- 
generation is  steadily  gaining  ground,  and  by  some  its  possible 
origin  even  from  the  albumins  is  denied.  On  the  other  hand, 
there  can  be  no  doubt  that  under  normal  conditions  fats  can 
originate  from  albumins.  This  has  been  shown  by  various  experi- 
ments. Large  quantities  of  fat  can  thus  be  isolated  from  the 
liver  of  dogs  which  have  been  poisoned  with  phosphorus  after 
having  previously  fasted  for  twelve  days.  In  such  an  event  it 
scarcely  seems  admissible  to  attempt  to  account  for  the  presence  of 
the  large  amounts  of  fat  in  the  liver  on  the  theory  of  a  transposi- 
tion. Bauer,  moreover,  has  pointed  out  that  while  in  such  cases  a 
largely  increased  elimination  of  nitrogen  occurs,  there  is  evidence  to 
show  that  a  non-nitrogenous  portion  of  the  albuminous  molecule  is 
retained,  as  the  absorption  of  oxygen  and  the  elimination  of  carbon 
dioxide  are  decreasetl  by  one-half. 

A  further  proof  of  the  possible  origin  of  fats  from  all)umins  has 
been    furnished    by    Hofmann.     Experimenting   with    maggots   of 


422  THE  SUPPOETISG   TISSUES. 

flies,  he  determined  the  amount  of  fat  in  one  portion  directly,  and 
then  permitted  a  second  portion  of  the  same  weight  to  develop  in 
defibrinated  blood  containing  a  known  amount  of  fat.  These  were 
then  killed  and  analyzed.  The  result  showed  that  they  contained 
an  amount  of  fat  which  was  from  seven  to  eleven  times  as  large 
as  the  total  amount  of  fat  in  the  blood,  plus  the  amount  which  they 
originally  contained. 

Of  the  manner  in  which  the  fat  originates  from  alliuminous 
material  our  knowledge  is  still  imperfect.  That  an  actual  libera- 
tion of  fatty  radicles  can  occur  directly  appears  unlikely,  as  we 
have  no  evidence  whatever  to  show  that  the  albuminous  molecule 
contains  radicles  with  more  than  six  or  nine  atoms  of  carbon.  We 
have  shown,  however,  that  glucose  and  glycogen  can  both  be 
derived  from  this  source.  The  question  hence  suggests  itself.  Is  it 
possible  that  the  formation  of  fats  from  albumins  takes  place  with 
the  intermediate  formation  of  carbohydrates  ?  As  a  matter  of  fact, 
there  is  evidence  to  show  that  this  may  occur,  as  the  possible  origin 
of  fats  from  carbohydrates  is  now  well  established.  This  trans- 
formation represents  one  of  the  most  important  synthetic  phenomena 
which  occur  in  the  animal  world,  and  is  of  the  nature  of  a  synthetic 
reduction  in  which  the  CHOH  groups  of  the  carbohydrates  are  trans- 
formed into  CH2  groups. 

Magnus-Levy  has  recently  suggested  that  the  natural  synthesis 
of  fats  from  carbohydrates  takes  place  by  way  of  lactic  acid  and 
acetic  aldehyde  in  such  manner  that  the  higher  fatty  acids  result 
through  repeated  condensation  of  acetic  aldehyde  or  croton  aldehyde 
groups  and  subsequent  reduction. 

Tiie  possible  origin  of  fats  from  carbohydrates  can  be  demon- 
strated in  various  ways.  Two  animals  from  the  same  litter  and 
approximately  of  equal  weight  are  starved  until  the  stored  fat 
has  mostly  disappeared.  The  one  is  then  killed  so  as  to  ascertain 
the  amount  of  albumins  and  fats  which  still  remains.  The  other 
animal  is  now  fed  with  a  definite  quantity  of  some  cereal,  the  con- 
tained amounts  of  albumins,  starch,  and  fat  of  which  are  known. 
The  feces  are  carefully  collected  and  the  amount  of  non-resorbed 
fat  and  albumins  ascertained.  After  a  variable  period  of  time  this 
animal  is  also  killed,  and  the  amount  of  albumins  and  fat  esti- 
mated. The  increase  in  the  amount  of  albumins  must,  of  course, 
be  referable  to  that  ingested,  while  the  increase  in  the  fat  may  be 
in  part  due  to  the  ingested  fat,  in  part  to  albumins,  and  in  part  to 
carbohydrates.  In  such  cases  it  has  been  found  that  the  amount  of 
both  albumins  and  fat  which  were  contained  in  tlie  food  are  by 
no  means  sufiicient  to  account  for  all  the  accumulated  fat,  so 
that  the  conclusion  is  unavoidable  that  a  certain  proportion  of  this 
must  be  referable  to  the  ingested  carbohydrates.  Calculation  has, 
indeed,  shown  that  as  much  as  86.7  per  cent,  of  the  fat  is  of  such 
origin. 

Significance  of  the  Fats. — As  regards  the  function  of  fat  in  the 


ADIPOSE  TISSUE.  423 

animal  body,  our  knowledge  is  very  imperfect.  Owing  to  its 
property  as  a  poor  conductor  of  heat,  it  is  probably  of  moment 
in  preventing  an  undue  irradiation.  Its  principal  significance,  how- 
ever, is  undoubtedly  connected  with  its  manifest  value  as  a  food-stuff 
and  as  a  source  of  energy.  But  we  do  not  know  whether  this  is 
expressed  in  any  specific  function  of  the  body  beyond  the  produc- 
tion of  heat  in  general.  As  the  fat  disappears  during  starvation 
before  the  albumins  are  attacked,  we  may  assume  that  its  presence 
under  normal  conditions  prevents  an  undue  destruction  of  those  ele- 
ments which  essentially  represent  the  living  tissue.  In  this  respect, 
however,  the  fats  are  inferior  to  the  carbohydrates,  as  is  apparent 
from  the  fact  that  in  starving  animals  the  administration  of  fats  does 
not  lead  to  so  marked  a  diminution  in  the  elimination  of  nitrogen 
as  is  effected  by  a  corresponding  amount  of  carbohydrates.  Upon 
this  basis  also  Voit  has  explained  the  well-known  phenomenon  that 
herl^ivorous  animals  are  more  likely  to  accumulate  albumins  than  the 
carnivora,  as  the  latter  receive  scarcely  any  carbohydrates  in  their 
food,  while  that  of  the  former  contains  comparatively  large  amounts. 


CHAPTER    XX. 

THE  SKIN  AND   ITS  APPENDAGES. 

In  considering  the  chemical  composition  of  the  skin  and  its 
appendages,  we  shall  deal  more  exclusively  with  those  substances 
which  are  more  or  less  peculiar  to  its  epidermal  structures.  The 
remaining  components  have  already  been  studied  in  detail  and 
require  no  further  consideration  at  this  place. 

The  epidermal  structures  in  the  case  of  the  vertebrate  animals 
comprise  the  epithelial  lining  of  the  skin,  together  with  its  sweat 
glands,  the  sebaceous  glands  and  allied  glands,  the  hair,  the  nails, 
the  hoofs,  the  feathers,  etc. 

On  section  of  the  epidermis  we  discern  diiferent  layers  of  cells. 
The  lowest  of  these,  which  is  known  as  the  Malpighian  layer,  is 
composed  of  the  youngest  cells,  from  which  all  others  are  derived. 
These  are  distinctly  protoplasmic  in  character,  but  with  increasing 
age  they  become  dry  and  scaly,  and  are  finally  represented  by  fine 
laraellse  of  keratin,  which  are  constantly  thrown  oft' and  regenerated 
from  below.  Keratin,  itself,  however,  is  not  found  in  the  lower 
layers  of  the  epidermis,  as  has  been  shown  by  Ernst,  but  appears 
only  above  the  so-called  stratum  granulosum.  In  the  latter  we 
find  peculiar  granules,  which  are  scattered  about  the  nuclei  of  the 
cells,  and  which  Ernst  regards  as  derivatives  of  the  nuclei.  They 
are  known  as  eleidin  granules,  and,  according  to  some  observers, 
represent  intermediary  products  in  the  formation  of  keratin.  Of 
their  chemical  nature,  however,  nothing  is  known. 

The  transition  of  the  soluble  albumins  of  the  lower  strata  of  cells 
into  the  insoluble  keratin  also  finds  its  expression  in  the.  greater 
resistance  which  the  upper  layers  offer  to  the  action  of  the  caustic 
alkalies.  For,  while  the  lower  cells  are  dissolved  with  comparative 
ease  the  upper  strata  are  scarcely  affected  by  the  reagent.  Pancreatic 
juice  and  gastric  juice  behave  in  the  same  manner,  and  it  is  thus 
possible  to  separate  the  insoluble  keratin  from  the  soluble  albumins 
which  may  be  present  at  the  same  time.  Like  the  horny  layer  of 
the  skin,  so  also  are  other  epidermal  structures,  such  as  nails, 
hair,  horn,  feathers,  etc.,  largely  composed  of  keratins,  and  it  is 
noteworthy  that  these  keratins  are  especially  rich  in  sulpluir.  That 
of  human  liair,  according  to  Suter,  contains  2.52  per  cent.,  of  which 
2.34  per  cent,  is  present  in  loosely  combined  form,  which,  as  we 
have  seen,  is  most  likely  represented  by  a  cystin  group.     Horn 

424 


THE  SKIN  AND  ITS  APPENDAGES.  425 

sliavings  contain  even  more,  viz.,  3.42,  of  which  2.53  is  loosely 
combined. 

In  addition,  we  find  a  variable  amount  of  salts,  among  which  the 
insoluble  forms  are  especially  important.  Their  presence  manifestly 
serves  the  purpose  of  increasing  the  rigidity  of  these  structures. 
Especially  noteworthy  is  the  large  amount  of  silicic  acid  which  is 
found  in  hair  and  in  feathers.  Besides  this,  we  meet  with  varial)le 
amounts  of  phosphates  and  sulphates  of  the  alkalies  and  alkaline 
earths,  and  very  curiously  also  with  inm  salts.  The  fact  that  larger 
amounts  of  the  latter  are  found  in  dark-colored  hair  than  in  hair 
of  a  lighter  color  suggests  that  their  presence  may  be  dependent 
upon  these  pigments.  Of  interest  is  the  fact  that  Gautier  was  able 
to  demonstrate  the  presence  of  traces  of  arsenic  as  a  constant  con- 
stituent of  the  skin  and  its  appendages,  viz.,  the  hair,  the  wool  of 
sheep,  etc. 

The  black  and  brown  pigments  which  are  found  in  the  hair  and 
in  the  skin  of  the  negro  belong  to  the  group  of  the  melanins. 
Individually  these  bodies  are  but  little  known,  and  it  is  an 
open  question  whether  the  iron  that  is  found  in  the  asli  is  present 
in  these  structures  in  molecular  combination  with  the  pigments. 
Unlike  the  fuscin  of  the  choroid  and  the  hippomelanin  that  has  been 
obtained  from  melanotic  tumors  in  horses,  the  melanins  of  the  skin 
and  the  hair  are  easily  soluble  in  solutions  of  the  alkaline  hydrates. 
They  contain  sulphur  (2  to  4  per  cent.),  but  not  in  so  large  amounts 
as  the  phyuiatorhusin  that  has  been  isolated  from  melanotic  gro\vths 
of  man  and  from  the  urine  (8  to  10  per  cent.).  Bodies  belonging 
to  this  order  apparently  have  also  been  encountered  among  the 
products  of  hydrolytic  decomposition  in  the  case  of  the  common 
albumins,  and  Pick  has  described  a  similar  substance  which  he  found 
in  Witte's  peptone,  and  which  he  designates  as  pej)tomelanin.  Con- 
jointly the  melanins,  which  can  be  obtained  from  the  albumins,  are 
termed  melanoidins  (Schmiedeberg).  On  fusion  with  potash  skatol 
and  indol  result ;  heated  in  the  dry  state  with  powdered  zinc  they 
give  an  intense  pyrrol  reaction  with  pine  wood  and  hydrochloric  acid. 
On  reduction  of  the  melanoidin  obtained  from  serum-albumin  with 
zinc  in  a  current  of  hydrogen,  as  also  from  the  melanin  of  the 
choroid,  and  that  of  melanotic  growths,  pyridin  has  been  obtained. 
In  addition  Samuely  found  a  supposedly  aromatic  body  with  a 
benzaldehy de-like  odor. 

As  regards  the  origin  of  the  melanoidins,  we  may  imagine  that 
the  various  chromogenic  groups  which  occur  in  the  albumins,  and 
which  contain  or  form  aromatic  (tyrosin)  and  mainly  heterocyclic 
radicles  (pyrrol,  pyridin,  skatol),  are  condensed  to  dark-colored 
products  on  boiling  with  acids,  with  coincident  loss  of  water  and 
taking  up  of  oxygen,  and  that  the  mixtures  of  these  products 
represent  the  melanoids. 

E.  Spiegler  seems  to  have  demonstrated  conclusively  that  the 


426  THE  SKIN  AND  ITS  APPENDAGES. 

pigment  of  black  hair  is  not  a  derivative  of  blood-pigment,  as  it  is 
impossible  to  obtain  either  hsemopyrrol  or  a  hajmatinic  acid  on 
appropriate  treatment  of  the  isolated  prodnct,  viz.,  substances 
which  must  be  regarded  as  characteristic  reduction-,  viz.,  oxidation-, 
products  of  the  blood-pigment.  On  oxidation  of  the  black  pig- 
ment of  horse-hair  he  obtained  a  substance  of  the  composition 
CiiH^-.O.,,  which  seems  to  be  identical  with  Butlerow's  methy-dibutyl- 
acetic  acid.  The  elementary  composition  of  the  black  pigment 
obtained  from  diiferent  sources  differs  somewhat ;  that  derived  from 
black  horse-hair  had  the  composition  C^oHsgNgSOig,  another  from 
black  sheep  wool  C4gHggN8SO20. 

From  white  hair  of  horses  and  sheep  he  obtained  white  pigments 
of  the  composition  C^^iHygNipSOjo  and  CgiHggNn.SOjo.  These  white 
pigments  apparently  represent  the  chromogens  of  the  corresponding 
black  pigments. 

Into  the  various  pigments  which  have  been  found  in  the  skin  of 
reptiles,  in  the  scales  of  fishes,  in  the  feathers  of  birds,  etc.,  it  is 
scarcely  necessary  to  enter  at  this  place.  They  partly  belong  to  the 
melanins,  partly  to  the  lipochromes  ;  others  are  classified  as  mela- 
noids,  while  still  others  are  closely  related  to  the  hemoglobins.  To 
a  certain  extent,  moreover,  the  colors  of  birds'  feathers  appear  to  be  of 
a  jjhysical  nature  and  referable  to  certain  phenomena  of  interference. 

In  the  invertebrate  animals  various  pigments  are  also  observed, 
btit  are  fjr  the  most  part  unknown.  The  keratin,  as  I  have  already 
stated,  is  here  represented  by  other  tegumentary  substances,  such  as 
chitin,  tunicin,  the  hyalogens,  and  the  skeletins  (which  see). 

The  Sweat. — The  sweat  is  the  specific,  secretory  product  of  the 
corresponding  glands,  which  are  found  imbedded  in  the  lower  portion 
of  the  dermis.  In  man,  these  glands  rank  next  in  order  to  the 
kidneys  in  importance  as  excretory  organs  of  water,  and  are  capa- 
ble, to  a  certain  extent  at  least,  of  assuming  a  vicarious  activity 
when  the  kidneys  are  diseased.  In  man  their  number  is  quite 
large,  exceeding  2,000,000.  Their  distribution,  how^ever,  is  not 
uniform,  and  as  a  result  there  are  certain  portions  of  the  body 
in  which  a  more  abundant  secretion  is  noted  than  in  others.  Such 
regions  are  the  forehead,  the  armpits,  the  palms  of  the  hands,  the 
soles  of  the  feet,  etc. 

Among  the  mammalian  animals,  however,  some  exist  in  which 
a  secretion  of  sweat  does  not  occur.  This  is  the  case  in  many 
of  the  rodents  and  the  goat.  The  sheep,  the  horse,  and  the  apes, 
on  the  other  hand,  sweat  over  their  entire  body,  while  other  animals, 
like  the  cat  and  the  dog,  sweat  only  from  the  balls  of  the  toes. 

The  amount  of  sweat  excreted  in  man  is  very  variable.  It  differs 
in  different  individuals  ;  and  is  dependent  upon  the  surrounding 
temperature,  the  amount  of  water  ingested,  the  temperature  of  the 
body,  the  amount  of  exercise  taken,  etc.  It  is  manifestly  under  the 
control  of  the  central  nervous  system,  and  is  increased  by  painful 


THE  SKIN  AND  ITS  APPENDAGES.  427 

sensations  and  emotions,  by  stimulation  of  the  sciatic  nerve,  follow- 
ing the  administration  of  pilocarpin,  etc. 

Ordinarily  the  secretion  of  sweat  is  scarcely  noticeable,  as  the 
droplets  evaporate  almost  as  rapidly  as  they  appear  on  the  sur- 
face of  the  skin.  But  even  so,  from  700  to  900  c.c.  of  water  are 
daily  eliminated  by  the  body.  Artificially  this  amount  can  be 
greatly  increased,  and  it  is  stated  that  from  <iOOO  to  8000  c.c.  may 
be  excreted  in  the  twenty-four  hours  if  the  body  is  kept  at  a  tem- 
perature of  from  40°  to  50°  C,  and  large  amounts  of  fluid  are 
ingested. 

Sweat,  recently  secreted,  is  more  or  less  turbid,  owing  to  an  admix- 
ture of  desquamated  epithelial  cells,  and  droplets  of  fat,  which 
are  in  part  derived  from  the  sebaceous  glands,  but  are  to  some 
extent  also  referable  to  the  sweat-glands  proper.  After  filtration  it 
appears  as  a  clear,  transparent,  colorless  fluid,  of  a  salty,  somewhat 
acrid  taste,  and  a  very  characteristic  odor,  which  differs  somewhat 
according  to  the  region  of  the  body  from  which  the  sweat  is  derived. 
Its  specific  gravity  varies  between  1.004  and  1.005. 

At  the  beginning  of  its  secretion  the  sweat  presents  an  acid  reac- 
tion, which  is  probably  referable  to  an  admixture  of  fatty  acids 
derived  from  the  sebaceous  glands  ;  later,  however,  it  is  alkaline. 

Under  normal  conditions  the  sweat  is  essentially  a  very  dilute 
aqueous  solution  of  mineral  salts,  but  in  addition  we  also  find  small 
amounts  of  many  urinary  components,  such  as  urea,  uric  acid, 
kreatinin,  aromatic  oxy-acids,  volatile  fatty  acids,  skatoxyl  and 
phenol  sulphate,  besides  fat,  cholesterin,  traces  of  albumin,  salts  of 
lactic  acid,  and  so-called  sudoric  or  hydratic  acid,  etc.  Larger 
amounts  of  solids  are  principally  met  with  in  cases  of  renal  insuffi- 
ciency, and  it  may  then  hap])en  that  the  elimination  of  urea  through 
the  sweat  increases  to  10  grammes,  as  compared  with  0.043-1.55 
pro  mille,  which  may  be  regarded  as  normal.  In  some  cases  of 
this  kind  the  urea  may  actually  be  found  in  the  form  of  a  fine  cry- 
stalline powder  deposited  all  over  the  skin.  Glucose  has  been 
observed  in  diabetes.  Cystin  has  been  noted  in  cases  of  cystinuria, 
and  abnormally  large  amounts  of  uric  acid  have  been  found  in 
gout.  In  jaundice  bilirubin  may  color  the  sweat  a  bright  yellow. 
A  blue  and  red  color,  which  is  thought  to  be  referable  to  the 
presence  of  indigo-blue  and  indigo-red,  has  also  been  observed 
(chromhidrosis  or  cyanhidrosis)  ;  and  it  is  stated  that  in  rare 
instances  blood  may  appear  in  the  sweat  (hsemahidrosis).  It  is 
noteworthy,  furthermore,  that  a  number  of  foreign  substances, 
when  ingested  by  the  mouth,  such  as  quinin,  the  various  salts 
of  iodine,  mercury,  and  arsenic,  are  in  part  eliminated  in  the 
S'vi'^eat. 

A  general  idea  of  the  quantitative  composition  of  the  sweat  may 
be  formed  from  the  accompanying  analyses,  which  are  taken  from 
Favre,  Sch5ttin,  and  Fiinke. 


428  THE  SKIN  AND  ITS  APPENDAGES 

Sweat  in  gen- 
eral (obtained  Sweat 
by  elevation        (from   extremities), 
of  tempera- 
ture). 
Favre.             Schottin.        Fiinke. 

Water 995.573  977.40         988.40 

Solids 4.427  22.60  11.60 

(Soluble  in  water : 

Sodium  chloride 2.230  "    3.6    | 

Potassium  chloride 0.244  .    .    i 

Alkaline  sulphates 0.012  \  \^l\ 

Alkaline  phosphates traces]  '       [  ^  gg 

Albuminates 0.005  j 

Insoluble  in  water,  but  soluble  in  acidu- 
lated water :  ' 

Earthy  phosphates traces  0.39  J 

Soluble  in  alcohol  : 

Alkaline  lactates 0.317  ~|  1         « 04 

Alkaline  sudorates 1.562  I  n  qa      „f  ,."u;'i 

TT  AAio  r  li.oO  -  ol  which 

Urea 0.043  |  i  ^^s  ,,^-1. 

Fats  and  fiitty  acids 0.014  J  J     -^-^'^  "'^e. 

Insoluble  in  water  and  alcohol : 

Epithelium traces  4.20  2.49 


Gases. — While  in  mammals  and  birds  the  respiratory  fimction  of 
the  skin  is  insignifieant  as  compared  with  that  of  the  lungs,  we  find 
that  in  the  amphibia  life  may  persist  for  quite  a  while  after  removal 
of  the  lungs,  and  that  during  this  time  oxygen  is  actively  taken  up 
from  the  air  and  carbon  dioxide  eliminated  in  turn.  If,  however, 
the  exchange  of  gases  is  impeded  or  prevented  by  covering  the 
skin  with  a  thin  layer  of  varnish,  death  rapidly  takes  place.  This 
also  occurs,  it  is  true,  in  some  of  the  smaller  mammals  which  have 
a  delicate  skin,  but  it  is  now  known  that  the  fatal  end  is  here  not 
referable  to  the  impairment  of  the  cutaneous  respiration,  nor  to  a 
retention  of  waste  products,  as  was  formerly  supposed,  but  to  a 
paresis  of  the  cutaneous  vasomotor  nerves  and  a  resulting  dilatation 
of  the  bloodvessels.  As  a  result  an  abnormally  increased  irradia- 
tion of  heat  occurs,  which  constitutes  the  direct  cause  of  death.  If 
this  is  prevented  by  placing  the  animal  in  a  warm  chamber  or  by 
surrounding  it  with  cotton  and  the  like,  recovery  may  take  place. 
In  the  larger  mammals,  including  man,  in  which  a  coarser  skin 
exists,  no  deleterious  effects  are  noted  even  after  ten  days. 

The  amount  of  carbon  dioxide  which  is  given  off  by  man  through 
the  skin  is  principally  dependent  upon  the  surrounding  temperature, 
and  varies  between  8.4  grammes  at  29°  to  33°  C,  and  28.8  grainmes 
at  38.5°  C. 

The  Sebum. — The  sebum  is  the  specific  secretory  product  of  the 
sebaceous  glands,  and  serves  the  purpose  of  a  lubricant.  Amounts 
sufficient  for  analytical  pur])oses  can  be  obtained  from  newly  born 
children,  in  which  the  secretion  constitutes  the  so-called  vernix  case- 
osa.  In  the  fresh  state  it  is  a  semiliquid,  oily  material,  in  which 
on  microscopical  examination  can  be  discerned  desquamitted  epithe- 
lial cells  in  various  stages  of  degeneration,  fat  droplets,  fatty  acid 
needles,   and  quite  constantly  also  plates    of   cholesterin.     Almost 


THE  SKIN  AND  ITS  APPENDAGES.  429 

immediately  on  exposure  to  the  air  it  solidifies  to  a  white,  tallow-like 
material.     Its  reaction  is  alkaline. 

The  most  important  constituents  of  the  sebum,  as  also  of  the 
related  secretion  of  the  uropyg-ian  gland  of  birds,  are  the  compound 
cholesterins  (see  page  80).  Their  presence  has  been  demonstrated 
on  the  feathers  and  bills  of  birds,  in  the  wool  of  sheep,  in  the  hair  of 
mammals,  on  the  spikes  of  porcupines,  etc. ;  and  as  these  bodies  are 
remarkably  resistant  to  the  influence  of  putrefactive  organisms,  it 
has  been  supposed  that  their  presence  on  the  skin  and  its  append- 
ages serves  the  purposes  of  protecting  the  exposed  portions  of  the 
body  against  bacterial  invasion. 

In  addition  to  substances  of  this  order,  the  sebum  contains  a  vari- 
able amount  of  fats,  fatty  acids,  soaps,  lecithins,  mineral  salts,  and 
at  least  two  albumins,  of  which  one  is  commonly  regarded  as  casein. 

Closely  related  to  the  common  sebum  of  the  sebaceous  glands  of 
the  skin  proper  is  the  secretion  of  the  preputial  gland — the  so-called 
smegma  praeputii  and  the  cerumen  of  the  ceruminous  glands  of  the 
external  ear.  In  addition  to  the  common  constituents  of  the  sebum, 
the  smegma  also  contains  certain  components  of  the  urine  and  their 
decomposition-products,  such  as  ammonium  soaps,  and  in  the  horse 
hippuric  acid,  benzoic  acid,  and  oxalate  of  lime.  Its  peculiar  odor 
in  man  is  no  doubt  due  to  the  presence  of  certain  fatty  acids.  In 
the  secretion  of  the  beaver — the  castorcum  of  the  shops — this  is 
thought  to  be  referable  to  a  phenol-like  body,  while  in  the  corre- 
sponding product  of  the  musk-deer  (;musc)  a  volatile  base  is,  accord- 
ing to  Wohler,  the  active  odorous  principle. 

The  cerumen  differs  from  the  common  sebum  in  containing  a 
very  considerable  projiortion  of  potassium  soaps.  In  addition,  we 
also  meet  with  a  peculiar  yellow  pigment,  which  has  an  exceedingly 
bitter  taste  ;  but  of  the  composition  of  this  nothing  is  known. 

In  the  skin  glands  of  certain  amphibia,  finally,  and  notably  the 
toad  and  the  salamander,  poisonous  substances  have  been  found 
which  in  their  ])hysiological  effect  closely  resemble  the  action  of 
digitalis  and  strychnin.  They  have  been  termed  bufidin  and  sala- 
mandrin,  respectively.  In  the  secretion  of  the  toad,  moreover, 
methyl-carbylamin  and  isocyan-acetic  acid  have  been 'found,  of  which 
the  former  is  especially  toxic. 


CHAPTER  XXI. 

THE  GLANDULAR  ORGANS. 

THE  LIVER. 

The  functions  of  the  liver,  as  is  apparent  from  a  survey  of  the 
foregoing  chapters,  are  manifold.  During  embryonic  life  the  organ 
is  intimately  concerned  in  the  ])roduction  of  red  corpuscles,  and  at 
this  time  already  manifests  its  function  as  an  excretory  organ  also 
in  the  production  of  bile.  After  birth  its  hsemapoietic  activity 
ceases,  but  it  continues  important  as  an  excretory  organ  through 
which  the  decomposition-products  of  hsemoglobin,  in  so  far  as  they 
are  not  retained  and  utilized  in  the  formation  of  new  corpuscles,  are 
eliminated  from  the  body  in  association  with  taurin,  glycocoll, 
cholesterin,  and  the  cholalic  acids.  At  the  same  time,  however,  the 
liver  is  the  seat  of  some  of  the  most  important  syntheses  which  occur 
in  the  animal  body,  and  in  which  both  anabolic  and  katabolic  prod- 
ucts of  the  metabolism  are  involved.  AVe  have  thus  seen  that  the 
greater  portion  of  urea  in  mammals,  and  of  uric  acid  in  birds  and 
reptiles,  is  here  produced,  and  we  have  also  pointed  out  that  certain 
aromatic  substances  which  are  formed  during  the  process  of  intes- 
tinal putrefaction  or  have  been  ingested  as  such  are  transformed 
in  the  liver  into  conjugate  sulphates  and  glucuronates  and  are  thus 
rendered  innocuous.  Still  other  substances,  moreover,  which  are  for- 
eign to  the  body,  such  as  various  metallic  salts  and  certain  alkaloids, 
are  here  removed  from  the  general  circulation  when  artificially  intro- 
duced, and  it  is  for  tliis  reason  also  that  the  hypodermic  injection 
of  such  substances  is  mucli  more  efficacious  than  their  administra- 
tion by  the  mouth.  The  subsequent  elimination  of  the  metallic  salts 
then  occurs  in  part  through  the  bile,  and  to  a  great  extent  also 
through  the  intestinal  epithelium.  The  alkaloids  are  similarlv  re- 
moved, and  also  appear  in  the  urine  in  a  more  or  less  modified  form. 

Formerly  it  was  supposed  tliat  the  retransformation  of  peptones 
into  native  albumins  occurred  in  the  liver,  but,  as  I  have  shown, 
this  is  not  the  case.  On  the  other  hand,  we  have  seen  that  the  carbo- 
hydrates after  their  transformation  into  monosaccharides  are  carried 
to  the  liver,  and  are  here  stored  in  the  form  of  glycogen  when 
an  immediate  demand  for  glucose  does  not  exist  on  the  part  of  tlie 
other  organs  and  tissues  of  the  body.  This  transformation  of  mono- 
saccharides into  glycogen  represents  one  of  the  most  important  syn- 
theses which  occur  in  the  animal  body,  and  it  is  interesting  to  note 
that  whereas  glycogen  on  decomposition  always  gives  rise  to  the  for- 
mation of  glucose,  the  liver  is  capable  of  transforming-  the  other 
monosaccharides  into  glycogen  as  well  (see  also  page  184.) 
430 


THE  LIVER.  431 

Of  the  forces  which  are  at  work  in  bringing  about  these  various 
changes  in  the  liver  we  know  very  little,  but  to  judge  from  recent 
observations  it  appears  that  certain  tissue  ferments  are  here  primarily 
concerned. 

The  reaction  of  the  living  liver-tissue  is  alkaline.  After  death, 
however,  it  becomes  acid,  and  there  is  reason  to  believe  that,  as  in 
the  case  of  the  muscle-tissue,  this  acid  reaction  is  essentially  refer- 
able to  the  formation  of  lactic  acid.  At  the  same  time  the  tissue 
becomes  opaque,  owing  to  a  coagulation  of  the  liver  albumins. 

A  general  idea  of  the  chemical  composition  of  the  liver  may  be 
formed  from  the  following  analyses,  which  are  taken  from  v.  Bibra  : 

Man.  Ox. 

Water 761.7  713.9 

Solids 238.3  286.1 

Soluble  albumins 24.0  23.5 

Albuminoids 33.7  62.5 

Fats 25.0  32,8 

Extractives 60.7  49.1 

Insoluble  portion 94.4  112.9 

The  mineral  salts,  according  to  v.  Bibra,  constitute  about  1  per 
cent,  of  the  fresh  tissue,  and  are  essentially  represented  by  the  phos- 
phates of  potassium  and  sodium  and  a  fairly  large  amount  of  iron. 
Traces  of  manganese,  copper,  and  lead  are  also  found. 

The  Albumins. — The  albumins  of  the  liver-tissue  have  been 
notably  studied  by  Halliburton  and  Plosz.  The  gland  was  freed 
from  blood  and  bile  by  transfusion  with  ice-water  containing  0.75 
per  cent,  of  common  salt.  The  tissue  was  then  cut  into  small  pieces 
with  cooled  knives,  frozen  and  placed  under  pressure.  On  thawing, 
an  alkaline  fluid  could  thus  be  obtained,  which  represents  the  liver- 
plasma.  In  this  fluid  a  glol)ulin  exists  which  coagulates  at  45°  C, 
and  is  regarded  by  Halliburton  as  being  possibly  identical  with  one 
of  his  cell-globulins.  It  can  be  digested  by  gastric  juice.  In  addi- 
tion a  nucleoproteid  was  found,  which  coagulated  at  70°  C,  and 
which  yielded  an  insoluble  residue  of  nuclein  on  digestion.  From 
the  cells  proper  they  extracted  a  globulin  with  a  10  per  cent,  solution 
of  sodium  chloride,  whicli  coagulated  at  75°  C,  and  which  may  also 
l:>e  identical  with  one  of  Halliburton's  cell-globulins ;  further,  an 
albumin  (coagulation-point  70°-73°  C.)  and  an  alkaline  albuminate. 
In  addition,  a  glucoproteid  has  also  been  demonstrated,  which  yields 
a  reducing  substance  on  boiling  with  dilute  mineral  acids,  and 
which  is  probably  of  a  mucinous  character  and  derived  from  the 
connective  tissue  of  the  organ.  According  to  \yohlgemuth,  the 
nucleoproteid  of  the  liver  contains  2.98  per  cent,  of  phosphorus, 
and  a  pentose,  which  he  identified  as  /-xylose. 

The  nuclei  finally  contain  nucleoproteids,  and  it  is  of  special 
interest  to  note  that  at  least  two  of  these  contain  iron.  The  one  is 
apparently  identical  with,  or  at  least  closely  related  to,  the  hcemato- 
gen  of  birds'  eggs,  while  in  the  other,  which  Zaleski  terms  hcpatln, 
the  iron  is  even  more  firmly  combined.     The  occurrence  of  these 


432  THE  GLANDULAR   ORGANS. 

iron-containing  products  is  important  in  view  of  the  fact  that  the 
iron  which  is  furnished  in  the  food  can  apparently  be  utilized  only 
by  the  body  in  the  formation  of  haemoglobin,  when  introduced  in 
such  forju.  It  has  hence  been  suggested  that  these  nucleins  after 
resorption  are  temporarily  deposited  in  the  liver  until  required  by 
the  hfemopoietic  organs. 

The  iron  which  is  present  in  the  liver  in  molecular  combination 
with  the  nucleoproteids  can  be  demonstrated  only  after  the  isolation 
of  the  nucleins  in  question  and  their  incineration.  But  in  addition 
we  also  find  iron  in  combination  with  albumins  as  so-called  iron- 
albaminates,  from  Avhich  the  metal  can  be  split  oif  by  treating 
with  acid  alcohol.  The  presence  of  this  form  can  be  directly 
demonstrated  by  moistening  a  slice  of  the  liver-tissue  with  hydro- 
chloric acid,  and  then  with  a  solution  of  potassium  ferrocyanide  or 
potassium  sulphocyanide,  when  a  blue,  viz.,  a  red  color  develops. 
As  regards  the  origin  of  these  iron-albuminates,  the  opinion  prevails 
that  thev  are  formed  within  the  tissues  of  the  body,  and  are  refer- 
able to  the  disintegration  of  red  corpuscles.  They  are  accordingly 
also  found  in  the  spleen  and  the  bone-marrow,  and  are  met  with 
in  increased  amounts  in  conditions  which  are  associated  with  an 
increased  destruction  of  red  corpuscles.  Large  quantities  are  thus 
especially  noted  in  cases  of  pernicious  anteraia,  in  acute  infantile 
gastritis,  and  in  poisoning  with  arsenious  hydride,  where  their 
presence  constitutes  the  phenomenon  of  so-called  siderosis  of  the 
liver.  According  to  Vay,  the  average  quantity  of  iron-albuminate 
which  can  be  isolated  from  the  fresh  organ  under  normal  conditions 
amounts  to  from  0.15  to  0.3  per  cent.,  corresponding  to  from  0.01 
to  0.018  per  cent,  of  iron.  The  total  amount  of  iron  which  is 
normally  found  in  the  liver-cells  during  adult  life  is  subject  to  con- 
siderable variation,  which  is  more  extensive  in  men  (0.048  to  0.367 
per  cent.)  than  in  women  (0.05  to  0.092  per  cent.).  On  an  average 
the  cells  of  women  contain  less  iron  than  the  cells  of  men.  The 
lowest  values  are  found  between  the  twentieth  and  the  twenty-fifth 
year. 

The  occurrence  of  especially  large  amounts  of  iron  in  the  liver  of 
newly  born  animals  is  probably  referable  to  the  hsemapoietic  activity 
of  the  organ  during  embryonic  life. 

Isolation  of  the  Iron-containing  Nucleins. — To  prevent  any  con- 
tamination with  hremoglobin,  it  is  necessary  to  remove  all  traces  of 
blood  from  the  liver.  To  this  eixl,  Bunge  has  suggested  the  follow- 
ino-  method  :  in  the  living;  animal  which  has  been  anaesthetized  with 
morphin  and  chloroform  a  cannula  is  tied  into  the  portal  vein. 
Through  this  a  stream  of  a  1  per  cent,  solution  of  sodium  chlo- 
ride heated  to  the  body  temperature  is  introduced  under  moderate 
pressure.  As  soon  as  the  solution  begins  to  flow  the  hepatic  artery 
and  the  hepatic  veins  are  divided  and  the  aV)domen  closed.  A 
minute  later  it  is  reopened,  the  liver  is  dissected  out  and  placed 
in  a  porcelain  bowl,  while  the  transfusion  is  continued.     The  bowls 


THE  LIVER.  433 

are  changed  until  perfectly  clear  saline  solution  flows  from  the  veins. 
To  attain  this  end,  the  transfusion  need  be  carried  on  for  only  a  few 
minutes.  If  successfully  performed,  the  liver  should  present  a  uni- 
formly light-brown  color,  and  a  portion  of  the  minced  organ  when 
placed  in  distilled  water  should  leave  this  entirely  uncolored.  The 
gall-bladder  is  now  removed,  the  organ  pressed  between  filter-paper, 
finely  hashed,  and  enveloped  in  muslin.  It  is  then  thoroughly 
kneaded  under  water.  The  connective  tissue  and  vessels  are  thus 
separated  from  the  cellular  elements  and  remain  behind.  The  cells 
are  thoroughly  extracted  with  water  and  with  dilute  saline  solution, 
by  decantation,  until  all  soluble  substances  have  been  removed. 
They  are  then  digested  with  gastric  juice.  The  non-digested  residue 
is  extracted  with  acidulated  alcohol  and  subsequently  with  ether,  to 
remove  pigments,  cholesterin,  and  fats.  It  is  then  treated  wuth 
weak  ammonia-water,  which  dissolves  the  iron-containing  nncleins. 
From  this  solution  they  are  })recipitated  with  absolute  alcohol  when 
added  in  excess.  The  resulting  material  constitutes  the  hepatin  of 
Zaleski.  The  other' iron-containing  nuclein  is  a})parently  present 
in  the  liver  as  a  nucleo-albumin,  and  is  found  in  this  form  in  the 
saline  extract  of  the  cells.  To  demonstrate  its  presence,  the  previ- 
ous extraction  Avith  saline  solution  is  omitted.  If  the  residue  of 
nucleins,  which  remains  after  digestion  with  gastric  juice,  is  then 
placed  in  a  solution  of  ammonium  sulphide,  a  greenish  color  gradu- 
ally develops  which  ultimately  turns  black,  owing  to  the  forma- 
tion of  sulphide  of  iron.  The  hepatin  itself  does  not  give  this 
reaction.  Neither  sul)stance  gives  up  its  iron,  even  when  treated 
with  acidulated  alcohol  ^  for  days,  thus  clifiering  from  the  iron  albu- 
minate, which  behaves  in  this  manner  exactly  like  inorganic  prep- 
arations of  iron. 

Isolation  of  the  Iron-containing  Albuminates. — In  this  case  it  is 
not  necessary  previously  to  wash  out  the  blood.  The  organ  is 
minced  w'ithout  further  preparation,  and  is  placed  in  from  three  to 
four  times  its  volume  of  water.  The  mixture  is  slowly  heated, 
boiled  for  about  fifteen  minutes,  and  filtered  on  cooling.  The  filtrate 
is  carefully  precipitated  with  a  10  per  cent,  solution  of  tartaric  acid. 
The  resulting  flocculent  material,  which  presents  a  brown  color,  is 
collected  on  a  filter,  washed  with  a  weak  solution  of  tartaric  acid, 
then  with  50  per  cent,  alcohol,  and  finally  with  absolute  alcohol.  It 
contains  about  6  per  cent,  of  iron.  It  is  soluble  in  solutions  of  the 
alkalies,  and  does  not  react  with  ammonium  sulphide  at  once.  After 
a  few  minutes,  however,  the  solution  becomes  darker  and  gradually 
turns  black.  On  treating  with  acid  alcohol  (see  above)  the  iron  is 
split  off,  and  can  be  directly  demonstrated  by  testing  with  potassium 
ferrocvanide  or  potassium  sulphocyanide.  Schmiedeberg  has  termed 
the  substance  in  question  ferratin,  and  regards  it  as  a  ferri-albuminic 
acid. 

'  The  acidulated  alcohol  contains  10  volumes  of  a  25  per  cent,  solution  of  hydro- 
chloric acid  and  90  volumes  of  96  per  cent,  alcohol  (Bunge's  fluid). 

28 


434  THE  GLANDULAR   ORGANS. 

Ferments. — Thus  far  the  following  tissue-ferments  have  been 
demonstrated  in  the  liver-cell :,  a  maltase,  a  glucase,  a  proteolytic 
ferment,  a  nuclease,  an  aldehydase,  a  laccase,  a  ferment  which  is 
capable  of  transforming  the  firmly  united  nitrogen  of  the  amido- 
acids  into  ammonia;  further,  a  fibrin-ferment,  one  which  effects  the 
transformation  of  glycogen  to  glucose,  and  probably  also  a  lipase, 
and  a  rennin-like  ferment. 

In  addition  the  liver  contains  a  substance,  possibly  a  proferment, 
which  when  activated  by  a  certain  princi])le  obtained  from  the 
pancreas  (a  kinase)  is  capable  of  causing  extensive  glucolysis.  To 
be  sure,  the  glucose  of  the  liver  disappears  of  itself  when  the  organ 
is  removed  from  the  body,  but  we  may  well  imagine  that  this  is 
owing  to  the  presence  of  a  certain  amount  of  the  kinase.  Liver- 
tissue  to  which  pancreas  is  added  will  hydrolyze  a  much  greater 
amount  of  glucose  than  liver-tissue  alone  can  do  (Hirsch).  The 
conditions  here  are  thus  quite  similar  to  what  takes  place  in  the 
carbohydrate  metabolism  of  muscle-tissue. 

Glycogen. — Amount. — The  amount  of  glycogen  which  occurs  in 
the  liver  is  primarily  dependent  upon  the  state  of  nutrition  of  the 
animal  and  the  amount  of  exercise  that  is  taken.  This  is  apparent 
from  the  fact  that  it  is  constantly  consumed  during  the  activity  of  the 
muscle-tissue  more  esjiecially,  but  is  also  utilized  in  the  regeneration 
of  all  cellular  elements  of  the  body.  During  starvation  it  rapidly 
disappears,  but  it  is  also  rapidly  formed  if  carbohydrates  are  then 
ingested.  Maximal  amounts,  according  to  Kiilz,  are  found  after 
from  fourteen  to  sixteen  hours  follo\ving  the  administration  of  food. 
It  has  been  calculated  that  in  the  liver  of  man  150  grammes  can  be 
stored  at  one  time.  This  would  correspond  to  about  10  per  cent, 
for  an  organ  weio^hing;  1500  o^rammes.  In  dogs  which  have  been 
fed  on  potatoes  and  bread  Pavy  claims  to  have  found  as  much  as  17 
per  cent.  After  death  the  transformation  of  glycogen  into  glucose 
continues  as  in  the  case  of  muscle-tissue,  and  in  order  to  ascertain 
the  exact  amount  which  was  present  during  life  it  is  hence  necessary 
to  remove  the  organ  at  once  and  to  prevent  the  further  inversion  of 
the  material,  by  the  living  protoplasm  or  the  contained  ferments,  by 
placing  the  tissue  in  boiling  water. 

Properties. — The  pure  substance  represents  a  white,  amorphous 
powder,  which  is  both  odorless  and  tasteless.  In  water  it  forms  an 
opalescent  solution,  from  which  it  can  be  precipitated  by  the  addi- 
tion of  alcohol,  after  adding  a  little  sodium  chloride,  or  by  means 
of  lead  subacetate.  The  substance  is  dextrorotatory.  The  specific 
deo:ree  of  rotation,  however,  seems  to  be  influenced  bv  various 
factors.  In  pure  solution  it  is  given  as  +  196.63°.  It  does  not 
reduce  Fehling's  solution,  but  can  maintain  cujn'ic  hydroxide  in 
solution.  After  the  addition  of  a  little  sodium  chloride  its  solutions 
are  colored  red  by  treating  with  iodine.  With  benzoyl  chloride,  in 
the  presence  of  sodium  hydrate,  it  gives  a  granular  precipitate  of 
benzoyl-glycogen.     On  boiling  with  dilute  mineral  acids  it  is  trans- 


THE  LIVER.  435 

formed  into  glucose.  Ferments  invert  it  to  maltose  or  glucose, 
accfjrding  to  the  nature  of  the  enzymes  at  "work.  ' 

Isolation  and  Quantitative  Estimation. — The  perfectly  fresh  liver, 
immediately  after  removal  from  the  animal,  is  placed  in  boiling 
water  and  divided  into  small  pieces.  After  boiling  for  a  few 
minutes  these  are  removed,  ground  to  a  pulp  with  sand  or  pulverized 
glass,  and  then  boiled  in  a  1  per  cent,  solution  of  sodium  hydrate, 
using  400  c.c.  for  every  100  grammes  of  tissue.  With  liver-tissue 
two  to  three  hours  suffice,  while  with  muscle-tissue  it  is  best  to  boil 
for  from  four  to  eight  hours.  Care  must  be  had  during  this  process 
that  the  concentration  of  the  alkali  does  not  exceed  2  per  cent. ;  to 
this  end  water  is  added  from  time  to  time.  The  alkaline  extract 
after  filtration  is  then  united  with  the  watery  solution  first  obtained, 
and  neutralized  with  hydrochloric  acid.  After  concentrating  the 
resulting  solution,  the  remaining  albumins,  notably  gelatin,  are  pre- 
cipitated on  cooling  by  alternate  treatment  with  a  solution  of  iodo- 
mercuric  iodide  and  hydrochloric  acid  added  drop  by  drop.  In 
the  filtrate  the  glycogen  is  precipitated  with  an  excess  of  alcohol. 
It  is  collected  on  a  filter,  washed  with  60  per  cent,  alcohol,  then 
with  absolute  alcohol  and  ether,  and  is  finally  dried  in  a  desic- 
cator over  sulphuric  acid.  From  the  weight  thus  obtained,  that  of 
the  combined  mineral  salts  must  be  deducted  after  incineration. 

Glucose. — The  amount  of  glucose  in  the  perfectly  fresh  liver 
varies  between  0.2  and  0.6  per  cent.,  but  rapidly  increases  at  the 
expense  of  the  glycogen  after  the  removal  of  the  organ  from  the 
body.  To  obtain  results  which  represent  the  actual  amount  that 
is  present  during  life,  it  is  hence  necessary  to  eliminate  the  inverting 
action  of  the  living  protoplasm  and  of  ferments  by  placing  the  organ 
in  boiling  water  immediately  after  the  death  of  the  animal.  It  is 
then  finely  minced,  thoroughly  extracted  with  boiling  water,  and  the 
sugar  determined  in  the  filtrate  according  to  the  usual  methods. 

Fat. — The  amount  of  fat  which  is  found  in  the  liver  is  quite 
large,  as  compared  Avith  the  other  organs  of  the  body,  and  normally 
varies  between  2  and  3.6  per  cent.  It  is  deposited  in  the  cells,  and 
beginning  along  the  periphery  of  the  acini  increases  in  amount  toward 
the  centre.  It  is  most  abundant  after  meals,  and  to  a  certain  degree  is 
dependent  upon  the  amount  of  fat  ingested.  Under  suitable  condi- 
tions the  infiltration  may  become  so  marked  as  to  simulate  fatty 
degeneration  ;  but,  in  contradistinction  to  fatty  infiltration,  we  find 
that  in  fatty  degeneration  the  amount  of  the  solids  is  markedly 
diminished.  The  amount  of  water  in  fatty  infiltration  is  diminished, 
while  in  degenerative  changes  it  is  perhaps  slightly  increased.  These 
relations  are  exemplified  by  the  following  figures,  which  are  taken 
from  Hammarsten  : 

water.  Fat.  ^^^^^ 

Normal  liver     .    .    .      770    pro  mille     20-35    pro  mille  207-195  pro  mille. 
Fattv  defeneration        816      "       "  87         "       "  97         "       " 

Fatt>  iniiltration     .  616-621  "       "       195-240    "       "       184-145    "       " 


436  THE  GLANDULAR   ORGANS. 

Extractives. — The  extractives  Avhieh  are  found  in  the  liver, 
aside  from  olycog^en  and  glucose,  are  notably  xanthin-bases,  which 
are  derived  from  the  nuclei.  They  comprise  xanthin,  hypoxanthin, 
guanin,  and  adenin.  Conjointly  they  represent  about  4.52  pro  mille 
of  the  dried  tissue.  They  can  be  isolated,  according  to  the  method 
described  on  page  391.  In  addition  we  find  small  amounts  of  urea, 
uric  acid,  paralactic  acid,  and  jecorin.  Cystin  also  has  been  isolated 
from  the  normal  liver  of  a  horse  and  from  that  of  the  porpoise,  but 
it  is  questionable  whetlier  the  substance  can  actually  be  regarded  as 
a  normal  constituent  of  the  gland.  It  has  once  been  obtained  from 
the  liver  of  a  patient  who  during  life  had  eliminated  cystin  in  the 
urine.  Under  pathological  conditions,  and  especially  in  acute  yellow 
atrophy,  large  quantities  of  paralactic  acid  have  been  found,  in 
addition  to  a  notable  amount  of  leucin  and  ty rosin.  In  amyloid 
degeneration  of  the  organ  chondroitin-sulphuric  acid  has  been 
observed.  Biliary  pigments  are  normally  not  encountered  in  the 
liver-cells,  but  quite  commonly  they  stain  these  an  intense  yellow  in 
cases  of  obstructive  jaundice.  These  various  constituents  have  been 
studied  in  the  foregoing  chapters,  and  need  not  be  reconsidered  at 
this  place. 

The  Digestive  Glands. 

Our  knowledge  of  the  chemical  composition  of  the  digestive 
glands,  viz.,  the  salivary  glands,  the  glands  of  the  stomach,  tlie  in- 
testinal mucosa,  and  the  pancreas,  is  largely  expressed  in  what  has 
been  said  regarding  their  specific  secretions.  With  the  exception  of 
the  pancreas,  the  cellular  elements  proper  have  not  been  studied  in 
detail. 

In  the  pancreas  a  highly  complex  nucleoproteid  occurs,  which  on 
boiling  with  water  yields  a  second  body  of  the  same  order,  which, 
however,  is  of  simpler  composition.  The  first  is  known  as  Ham- 
marsten's  nucleoproteid-a,  the  second  as  the  corresponding  -ft  prod- 
uct. Elementary  analvsis  of  this  ft  product  has  given  the  following 
results  :  C  =  34.0  ;  H  =  5.0  ;  N  ==  0.7  ;  P  =  4.5.  In  addition  the 
substance  contains  a  considerable  amount  of  iron.  Both  products 
contain  a  pentose  group  ;  from  the  a-  body  Grund  obtained  6.25  per 
cent.,  and  from  the  derived  body  amounts  varying  between  9.2  and 
15.4  per  cent. 

On  digestion  with  pepsin-hydrochloric  acid  a  nuclein  remains 
behind  which  is  very  rich  in  phosphorus.  This  yields  a  nucleinic 
acid,  which  Bang  has  termed  (juanyUc  acid  from  the  fact  that  on 
decom])osition  only  one  purin  base,  guanin,  is  obtained. 

Isolation  of  Guanylic  Acid  (Bang). — 1000-1200  grammes  of 
pancreas  (from  the  ox)  are  finely  hashed  and  suspended  in  two  liters 
of  1  per  cent,  sodium  hydrate  solution.  After  standing  for  twenty- 
four  hours  the  mixture  is  heated  until  it  ])ecomes  a'  thin  liquid  ; 
acetic  acid  is  then  added  until  the  reaction  is  distinctly  acid.  Tiie 
brownish-black  precipitate  which  results  is  collected  and  boiled  out 


THE  LYMPH-GLANDS.  437 

with  water.  Filtrate  and  washings  are  filtered,  rendered  feebly 
alkaline  with  ammonia,  concentrated  to  abont  300  c.c,  and  while 
still  hot  treated  with  3  volumes  of  alcohol.  The  resulting  precipi- 
tate is  filtered  oif,  dissolved  in  150  c.c.  of  water,  and  filtered  while 
hot.  On  cooling,  the  solution  is  again  precipitated  with  3  volumes 
of  alcohol.  This  process  is  repeated  once  more,  when  the  guanylic 
acid  is  obtained  in  pure  form.  1000  grammes  of  pancreas  yield 
about  3  grammes  of  the  acid. 

Tlhe  ferments  of  the  pancreatic  juice  have  already  been  considered. 
In  addition  we  find  various  autolytic  ferments,  and,  as  I  have 
pointed  out,  the  pancreas  (possibly  the  cells  composing  the  areas  of 
Langerhans)  also  furnishes  a  body,  which  may  belong  to  the  group 
of  kinases,  and  which  activates  certain  glucolytic  ferments  of  the 
liver  and  the  muscle-tissue. 

The  Lymph-glands. 

The  lymph-glands  comprise  the  lymph-glands  proper,  the  thy- 
mus gland,  and  the  spleen.  Their  fibrous  network  consists  essen- 
tially of  rcticidin,  but  also  contains  fibres  of  collagen  and  elastin. 
The  cellular  elements  have  been  studied  especially  in  the  case  of  the 
thymus.  They  contain  small  amounts  of  albumins,  lecithins,  fats, 
cholesterins,  traces  of  glycogen,  succinic  acid,  and,  according  to 
Kossel  and  Lilienfeld,  large  amounts  of  so-called  niicleohision. 
Lilienfeld's  nucleohiston,  according  to  the  researches  of  Huiskamp, 
Malengreau  and  Bang  is,  however,  no  unity,  but  a  mixture  of  two 
nucleoproteids,  viz.,  nucleohiston  and  a  nucleoproteid  which  con- 
tains no  histon  group.  The  nucleohiston  further  is  a  double  com- 
pound of  a  nucleinate  of  histon  and  a  pnranucleinate  of  histon — 
6  parts  of  what  Bang  terms  normal  acid-histon  (the  normal  acid 
being  an  adenin-guanylic  acid)  and  3  parts  of  adenylic  parahiston. 
The  difference  in  Bang's  concept  of  the  nucleohiston,  as  compared 
with  that  of  Kossel  and  Lilienfeld,  is  shown  below  : 

Nucleohiston  (Kossel)  Nucleohiston  (Bang) 

Histon  Leuconuclein  Histon  nucleinate        Histon  paranucleinate 

Albumin         Nucleinic  acid 

In  the  thymus  the  histon  (viz.,  parahiston)  nucleinate  represents  20 
per  cent,  of  the  total  weight,  as  compared  with  7  per  cent,  of  the 
second  nucleoproteid.  In  the  h/mph-glands  the  same  amount  of 
nucleoproteid  approximately  is  present,  but  only  5  per  cent,  of  the 
nucleinate  of  histon.  In  the  spleen  still  less  is  found,  and  the  bone- 
marrow  probably  contains  none. 

In  the  spleen  uric  acid  has  also  been  met  with,  and  as  in  the 
liver  iron-albuminates  occur,  which  may  be  isolated  as  there  de- 
scribed.    In  addition  small  amounts  of  inosit,  jecorin,  and  cerebro- 


438  THE  GLANDULAR   ORGANS. 

sides  have  been  encountered.     Giilewitsch  further  demonstrated  that 
arginin  is  a  normal  constituent  of  the  spleen. 

Of  ferments  a  proteolytic  ferment  has  been  discovered  in  the 
spleen  and  in  the  thymus  gland  by  Kutscher,  Conradi  and  Row- 
land. 

THE   KIDNEYS. 

In  addition  to  the  common  albuminoids  which  enter  into  the  com- 
position of  the  supporting  tissue  of  the  kidneys,  we  find  the  common 
extractives,  viz.,  nucleinic  bases,  uric  acid,  urea,  leucin,  inosit,  gly- 
cogen, fats,  and  at  times  taurin.  All  these  substances,  however,  are 
present  in  only  small  amounts.  On  one  occasion,  in  the  ox,  cystin 
has  also  been  encountered,  but  it  is  questionable  whether  this  is  a 
constant  constituent  of  the  organs.  Of  albumins,  Halliburton  has 
isolated  a  globulin  and  a  nucleoproteid,  with  coagulation-points  of 
52°  C.  and  (33°  C,  respectively.  In  addition,  a  mucin-like  body 
has  been  found,  which  does  not  yield  a  reducing  substance,  however, 
on  boiling  with  mineral  acids,  and  which  is  probably  a  nucleopro- 
teid. It  is  notably  found  in  the  papillary  portion  of  the  kidneys, 
while  the  other  nucleoproteid  is  principally  met  with  in  the  cortical 
portion.     Serum-albumin  is  said  to  be  absent. 

THE   MAMMARY   GLANDS. 

The  chemical  composition  of  the  mammary  glands  has  not  been 
studied  in  detail.  AVe  know,  however,  that  the  protoplasm  of 
the  functionally  active  glands  is  rich  in  albumins,  and  it  apjx'ars 
that,  as  in  the  case  of  the  pancreas^  a  very  complex  nucleo-gluco- 
proteid  is  here  also  present,  and  is  probably  intimately  concerned  in 
the  formation  of  tAvo  of  the  most  important  constituents  of  the  milk, 
viz.,  the  casein  and  lactose.  It  may  be  obtained  in  solution  by  first 
washing  the  gland  thoroughly  in  water,  so  as  to  free  it  from  milk  ; 
it  is  then  extracted  with  a  0.5  pro  mille  solution  of  sodium  hydrate 
at  ordinary  temperatures.  Such  solutions  also  contain  the  common 
albumins,  and  represent  an  exceedingly  viscid,  stringy  fluid,  from 
which  the  proteid  in  question  can  be  precipitated  by  acidifying  care- 
fully with  dilute  acetic  acid.  On  boiling  with  dilute  acids  the  sub- 
stance is  decomposed  into  albumin,  ph()S])h"oric  acid,  and  a  reducing 
substance  of  unknown  composition.  On  digestion  with  gastric  juice 
it  yields  a  paranuclein. 

As  in  the  case  of  the  pancreas,  the  substance  is  decomposed  by 
boiling  the  gland  with  water.  A  coagulable  albumin  and  a  nucleo- 
glucoproteid,  which  is  somewhat  less  complex  than  the  original  sub- 
stance, thus  result.  From  this  solution  the  proteid  can  be  precipi- 
tated by  the  addition  of  a  dilute  acid.  Like  its  mother-substance, 
it  also  yields  a  reducing  substance  on  hydrolytic  deeomposition. 
Of  the  relation  of  the  latter  to  lactose  nothing  is  known,  but  it  is 
noteworthy  that  this  is  formed  on   standing  if  a  functionally  active 


THE  MILK.  439 

gland,  wliile  perfectly  fresh,  is  ground  to  a  pul]^  and  kept  in  normal 
salt  solution  at  the  temperature  of  the  body.  An  intermediary  prod- 
uct is  then  also  apparently  formed,  \yliich  is  of  a  colloid  nature,  but 
not  identical  \yith  glycogen.  In  yie\y  of  recent  researches,  which 
tend  to  show  that  the  reducing  group  Avhich  is  present  in  the  gluco- 
proteids  is,  in  the  case  of  the  mucins  at  least,  not  a  true  carbohydrate, 
but  of  the  nature  of  chondroitin-sulphuric  acid  or  an  allied  substance, 
it  would  be  exceedingly  interesting  to  ascertain  whether  the  reduc- 
ing substance  in  the  case  of  the  mammary  nucleo-glucoproteid  also 
may  not  be  of  this  order. 

Of  other  constituents  of  the  gland,  we  find  yarious  xanthin- 
bases,  and  in  the  fimctionally  actiye  organ  also  a  certain  amount  of 
fat  which  is  present  in  the  form  of  globules  of  variable  size,  in  the 
bodies  of  the  cells. 

The  specific  secretory  product  of  the  mammary  glands  is  the  milk. 

The  Milk. — The  milk  is  the  specific  secretory  product  of  the 
mammary  glands,  and  constitutes  the  natural  food  of  all  mammals 
in  the  early  stages  of  their  extra-uterine  existence.  It  contains  all 
those  f  )od-stuflPs  which  are  necessary  for  the  maintenance  of  life, 
viz.,  albumins,  carbohydrates,  and  fats.  The  nutrient  components 
of  the  milk,  however,  are  more  or  less  specific  of  the  secretion 
in  question,  and  are  not  found  elsewhere  in  the  body  as  such. 
They  are  produced  in  the  gland  itself  from  the  common  constituents 
of  the  blood.  Among  these  the  albumins  are  the  most  important, 
and  there  can  be  little  doubt  at  the  present  time  that  the  fats  of 
the  milk  also  are  largely  referable  to  this  source.  This  is  appar- 
ent from  the  fact  that  in  the  bitch,  for  example,  the  amount  of  fat 
increases  with  an  increased  ingestion  of  meat  that  is  fi'ee  from  fats, 
while  it  is  diminished  when  the  animal  is  fed  on  fits  only.  The 
so-called  milk-sugar  also  is  apparently  derived  from  albumins,  as 
the  substance  continues  to  be  formed  although  no  carbohydrates  are 
ingested.  Its  amount,  however,  is  then  somewhat  smaller,  and 
increases  if  cane-sugar  or  starch  is  added  to  the  diet. 

General  Characteristics. — Fresh  milk  is  an  opaque,  white,  yellow- 
ish-white, or  bluish-white  liquid,  of  a  somewhat  creamy  consistence, 
a  more  or  less  sweetish  taste,  and  an  insipid  odor  which  is  peculiar 
to  the  particular  animal  from  which  the  milk  has  been  obtained. 
On  microscopical  examination  it  is  seen  that  the  opacity  is  largely 
due  to  the  presence  of  fat-globules,  which  vary  from  0.0024  to 
0.0046  ram.  in  diameter,  and  number  from  200,000  to  5,000,000 
per  cbmm.,  with  an  ayerage  of  about  1,050,000.  The  fat  is  thus 
present  in  a  state  of  fine  emulsion,  but,  in  contradistinction  to  other 
emulsions,  in  feebly  alkaline  media,  it  cannot  be  extracted  by  shak- 
ing with  ether  directly,  or  at  least  only  Avith  much  difficulty.  If,  on 
the  other  hand,  an  acid  or  a  caustic  alkali  is  previously  added  to  the 
milk,  this  is  readily  accomplished.  From  this  observation  it  has 
been  concluded  that  each  fat-globule  is  surrounded  by  an  albumi- 
nous membrane,  the   haptogenlc  membrane  of  Ascherson,  which  is 


440  THE  GLANDULAR    OBGAXS. 

dissolved  by  acids  and  alkalies,  but  which  normally  prevents  the 
solvent  action  of  the  ether  upon  the  contained  fat.  Later  investi- 
gations have  rendered  this  view  improbable,  however,  and,  as  a 
matter  of  fact,  no  one  has  ever  succeeded  in  demonstrating  the  jn-es- 
ence  of  a  special  membrane.  The  normal  resistance  to  the  action  of 
ether  is  now  explained  upon  the  assumption  that  each  globule  is 
surrounded  by  a  delicate  layer  of  albumin,  which  does  not  consti- 
tute a  true  membrane,  however,  but  is  formed  as  a  result  of  molecu- 
lar attraction.  It  is  possible,  indeed,  to  prepare  emulsions  of  fat 
artificially  by  shaking  with  albuminous  solutions,  which  in  their 
beliavior  to  ether  are  quite  similar  to  milk.  As  regards  the  char- 
acter of  the  particular  albumin  which  forms  this  layer,  our  kno\\'l- 
edge  is  not  complete.  It  has  been  supposed  by  some  that  it  is 
formed  by  casein,  but  there  are  reasons  for  believing  that  the 
albumins  of  the  milk  in  general  may  here  be  concerned. 

On  standing,  the  greater  portion  of  the  fat  rises  to  the  surface  of 
the  milk  and  forms  its  cream.  On  beating  the  milk  for  some 
time,  the  individual  fat-globules  are  caused  to  coalesce,  and  sepa- 
rate out  as  a  semisolid  mass,  which  constitutes  the  butter.  The 
remaining  liquid  is  termed  buttermilh,  and  still  contains  a  consider- 
able amount  of  fat  which  has  remained  in  emulsion. 

Besides  the  fat-glol)ules  the  milk  contains  also  innumerable  gran- 
ules of  calcium  phosphate  (probably  a  mixture  of  dij)hosphates  and 
triphosphates)  in  suspension,  which  are  visible  only  on  microscopical 
examination  and  are  said  to  number  about  4,000,000  per  cbmm. 
On  filtration  through  a  Chamberlain  filter,  under  pressure,  these 
remain  behind  together  with  the  fat.  But  we  then  also  find  that 
one  of  the  most  important  albuminous  constituents  of  the  milk, 
viz.,  casein,  Avhich  is  found  in  combination  with  lime,  is  likewise  not 
present  in  solution,  and  is  thus  obtained  in  the  form  of  a  thin,  jelly- 
like material.  The  filtrate  constitutes  the  milk-serum  and  contains 
tliose  components  of  the  fluid  which  are  present  in  a  state  of  actual 
solution. 

Upon  the  addition  of  chymosin  to  fresh  milk,  at  the  temperature 
of  the  body,  it  coagulates  almost  at  once.  The  resulting  clot, 
which  constitutes  cheese,  then  contracts  and  a  yellowish  fluid  gradu- 
ally appears,  which  is  termed  siceet  ichey.  During  this  process  the 
reaction  of  the  milk  is  not  changed.  A  similar  coagulation  is  noted 
when  fresh  milk  is  allowed  to  stand  exposed  to  the  air.  In  this  case, 
however,  the  reaction  of  the  whey  is  acid,  owing  to  the  formation  of 
lactic  acid  from  lactose  in  consequence  of  the  activity  of  certain 
micro-organisms. 

Perfectly  fresh  milk  does  not  coagulate  on  boiling,  but  it  will  be 
noted  that  a  skin  f  jrms  on  the  surface  of  the  milk,  which  is  rapidly 
reformed  when  removed.  This  consists  of  coagulated  casein  in  com- 
bination with  mineral  salts,  and  especially  phosphates  of  calcium. 
Actual  coagulation  does  not  occur,  even  if  a  current  of  carbon  dioxide 
has  previously  been  passed    through   the  liquid.     If  the  milk  has 


THE  MILK.  441 

stood  for  some  time,  however,  and  lactic  acid  fermentation  has  begun, 
a  tendency  to  coagulation  soon  becomes  manifest,  and  at  different  stages 
this  maj  then  be  effected  by  boiling  after  saturation  with  carbon 
dioxide,  then  by  boiling  alone,  subsequently  on  treating  with  carbon 
dioxide  without  boiling  ;  and  finally,  as  I  have  stated,  it  occurs 
spontaneously.  Sterilization  of  the  milk,  with  the  subsequent  exclu- 
sion of  micro-organisms,  as  also  the  addition  of  preservatives,  such 
as  boric  acid,  salicylic  acid,  thymol,  etc.,  will  prevent  lactic  acid  fer- 
mentation, and  consequently  also  coagulation  referable  to  this  source. 

On  exposure  to  the  air,  milk  is  said  to  absorb  its  own  volume  of 
oxygen  within  three  days. 

Amount. — -The  amount  of  milk  furnished  in  the  twenty-four  hours 
is,  of  course,  different  in  different  animals.  It  is  largely  dependent 
upon  the  development  of  the  glands,  and  accordingly  is  most  abun- 
dant in  those  animals  in  which  by  artificial  selection  a  marked  hyper- 
trophy of  the  organs  has  been  produced.  Some  cows  may  thus  yield 
24  liters  of  milk  in  tlie  tvventy-f  )ur  hours.  The  amount  is  further 
influenced  by  the  age,  as  also  by  the  character  of  the  diet,  the  amount 
of  liquid  ingested,  etc.  Especially  important  is  the  character  of  the 
diet,  and  notably  the  amount  of  albuminous  food  that  is  ingested. 
Where  this  is  deficient  the  amount  of  milk  is  diminished,  while, 
cceteris  paribus,  larger  amounts  are  furnished  if  an  abundance  of 
albumins  is  ingestetl. 

Women  furnish  from  900  to  1000  grammes  on  an  average  during 
the  height  of  lactation  ;  1500  grammes  probably  represent  the  maxi- 
mum output.  Good  cows  commonly  yield  from  6  to  10  liters,  goats 
and  sheep  about  1  liter,  in  the  twenty-four  hours.  With  the  gradual 
cessation  of  lactation  and  the  coincident  atrophy  of  the  mannnary 
glands  the  amount  decreases,  until  finally  the  secretion  is  arrested 
entirely.  In  women  and  cows  the  period  of  lactation  usually  lasts 
about  ten  months. 

Specific  Gravity. — Tlie  specific  gravity  of  the  milk  is  largely  de- 
pendent upon  the  amount  of  fat  present,  and  is  much  the  same 
in  different  animals.  Its  normal  variations  are  seen  in  the  accom- 
panying table : 

Woman 1.028-1.034 

Cow 1.029-1.034 

Goat        1.030-1.034 

Sheep 1.037-1.040 

Ass      1.029-1.035 

Mare      1.02.S-1.034 

Bitch      1.034-1.040 

Skimmed  milk  is,  of  course,  specifically  heavier  than  full  milk, 
and  a  higher  specific  gravity  is  accordingly  also  noted  in  milk  which 
is  poor  in  fat  than  in  rich  milk. 

Reaction. — Woman's  milk  and  that  of  most  herbivorous  animals 
is  uniformly  amphoteric,  owing  to  the  presence  of  diacid  and  mon- 
acid  phosphates  in  association  with  the  calcium  compoimd  of  casein. 
The  relative  values  of  the  acid  and  basic  components  in  cows'  milk 


442  THE  OLANDULAB   ORGANS. 

and  Imman  milk  are  given  below  in  terms  of  decinormal  sodium 
hydrate  and  .sulphuric  acid  solution.  The  figures  have  reference  to 
100  c.c.  of  milk  and  are  average  values  (Courant): 

iVNaOH  t'oHsSOi         Ratio. 

Hnman  milk 10.8  c.c.  3.6  c  c.         3:1 

Cows'   milk 41.0  c.c.         19.5  c.c.         2  :  1 

Human  milk  is  thus  relatively  more  alkaline  than  cows'  milk,  but 
is  absolutely  both  less  alkaline  and  less  acid. 

Mares'  milk  is  alkaline  and  that  of  the  carnivorons  animals  acid. 

Chemical  Composition. — A  general  idea  of  the  chemical  composi- 
tion of  the  milk  of  different  animals  and  of  woman  may  be  formed 
from  the  following  analyses,  which  are  taken  from  Konig,  Gorup- 
Besanez,  Hoppe-Seyler,  and  others  : 

Human.  Cow. 

Water 872.40-892.90  842.8-860.0 

Solids 108.00-127.60  140.0-157.2 

Albumins  (total)        16.13-36.91  33.0-43.2 

Albumin  (proper)      3.50-     9.91  1.2-     2.8 

Casein      12.80-  27.00  30.2-  42.0 

Fats      25.60-  43.20  40.0-  64.7 

Lactcse 53.90-  60.90  43.4-  50.0 

Salts 1.650-  4.200  6.3-     7.1 

A  survey  of  this  table  thus  shows  that  human  milk  contains  a 
smaller  amount  of  albumins  and  fats  but  more  lactose  than  cows' 
milk. 

In  addition  to  the  above  components  the  milk  contains  traces  of 
urea,  kreatin,  kreatinin,  hypoxanthin,  chole.sterin,  animal  gum,  and, 
curiously  enough,  citric  acid,  which  is  present  as  a  calcium  salt  to 
the  extent  of  from  0.18  to  0.25  per  cent.  Besides  these,  we  find  a 
small  amount  of  lecithins  and  a  yellow  lipochrome. 

Goat.  Sheep.  Marc.  Ass.  Dog.  Cat. 

Water 869.1  835.0  900.6  900.0  754.4  816.3 

Solids 130.9  165.0  99.4  100.0  245.6  18.3.7 

Albumins    .    .  36.9  57.4  1S.9  21.0  99.1  90.8 

Fats      ....  40.9  61.4  10.9  13.0  95.7  33.3 

Lactose    .    .    .  445  39.6  66.5  63.0  31.9  49.1 

Salts     ....  8.6  6.6  3.1  3.0  7.3  5.8 

Analysis  of  the  inorganic  components  of  human  milk  has  given 
the  following  results  (Bunge) :  the  figures  of  the  fir.st  column  were 
obtained  at  a  time  when  but  little  .^^odium  chloride  was  ingested, 
while  those  of  the  second  column  were  gotten  Avhile  the  woman 
ingested  30  grammes  a  day  (the  total  ash  is  calculated  as  1000  parts 
by  weight) : 

I.  II. 

Potassium  (KjO) 0.780  0.703 

Sodium  (Na.,Oj      0.232  0.257 

Calcium   (CaO) 0.328  0.343 

Magnesium   (MgO) 0.064  0.065 

Iron  (Fe^Oa) 0.004      '  0.006 

Phosphoric  acid  (P2O5) 0.473  0.469 

Chlorine  (CI) 0.438  0.445 


THE  MILK.  443 

The  differences  which  exist  in  the  composition  of  full  milk,  as 
compared  with  skimmed  milk,  cream,  buttermilk,  and  whey,  are 
shown  below  : 

•     ^cow'^').''  "^mTl?"^  Creaxn.  Buttermilk.        Whey. 

Water 871.7  906.6  655.1  902.7  932.4 

Solids 128.3  93.4  344.9  97.3  67.6 

Albumins     .    .  35.5  31.1  35.5  35.5  8.5 

Fats       ....  36.9  7.4  267.5  9.3  2.3 

Lactose      .    .    .  48.8  47.5  35.2  37.3  47.0 

Lactic  acid   .    .  none  none  none  3.4  3.3  . 

Salts 7.4  7.4  6.1  6.7  6.5 

Of  gases,  milk  contains  a  small  amount  of  oxygen  and  nitrogen, 
and  from  5.8  to  7.5  per  cent,  of  carbon  dioxide,  which  can  be 
removed  with  the  exhaust  pump. 

The  Albumins. — The  albumins  which  are  found  in  milk  are  casein, 
lactalburain,  and  so-called  lactoglobulin,  which  is  probably  identical 
with  the  serum-globulin  of  the  blood-plasma.  Of  these,  casein  is 
the  most  abundant  and  the  most  important. 

Casein. — Casein  is  a  nucleo-albumin,  and  has  the  character  of 
a  dibasic  acid.  In  the  dry  state  it  occurs  as  a  white  amorphous 
powder,  which  is  almost  insoluble  in  water,  in  dilute  acids,  and  solu- 
tions of  the  neutral  salts.  In  dilute  solutions  of  the  alkaline  hydrates 
and  in  lime-water  it  dis.solves  with  ease,  at  the  same  time  forming 
salts.  Such  solutions  are  neutral  or  slightly  acid  in  reaction,  accord- 
ing to  the  amount  of  alkali  that  has  been  added,  which  is  owing  to  the 
formation  of  neutral  or  acid  salts,  respectively.  When  triturated  in 
water  with  calcium  or  sodium  carbonate,  the  carbonates  are  decom- 
posed with  the  liberation  of  carbon  dioxide ;  the  same  salts  are  then 
formed  as  in  the  case  of  the  alkaline  hydrates.  Soldner*has  isolated 
two  calcium  salts  of  casein,  containing  1.55  and  2.36  percent,  of 
calcium  oxide ;  according  to  Courant,  these  are  dicalcium  and 
tricalcium  casein,  respectively.  The  salts  of  casein  with  the  alkalies 
and  alkaline  earths  are  readily  soluble  in  water,  even  in  the  absence 
of  neutral  salts,  and  are  hence  not  precipitated  on  dialysis.  On 
decomposition  with  dilute  acids  the  free  casein  is  obtained  again  in 
insoluble  form.  Suspended  in  water,  the  substance  is  coagulated  on 
boiling,  and  can  then  no  longer  be  dis.solved  without  undergoing 
denaturization,  as  on  boiling  with  acids  and  alkalies.  Solutions  of 
the  casein  salts,  on  the  other  hand,  do  not  coagulate  on  boiling,  but 
form  a  surface  skin,  as  in  the  case  of  milk.  The  salts  can  be  pre- 
cipitated from  their  solutions  by  salting  with  sodium  chloride  or 
magnesium  sulphate  to  saturation.  Metallic  salts,  such  as  copper 
sulphate,  also  precipitate  a  neutral  solution  completely. 

In  the  milk  the  casein  exists  as  a  neutral  lime  salt,  but  does  not 
occur  in  a  state  of  actual  solution,  as  has  been  pointed  out.  On 
filtering  milk  through  a  Chamberlain  filter  under  pressure  it 
remains  behind,  together  with  the  fat  and  calcium  phosphate,  as 
a  jelly-like    material.     On    treating   milk    with   a  dilute  acid    the 


444  THE  GLAXDULAE   ORGAXS. 

casein  is  precipitated,  as  in  the  case  of  the  aqueous  solution  of  its 
salts.  To  a  certain  extent  this  may  (jccur  in  the  stomach,  providing 
that  a  sufficient  amount  of  free  hydrochloric  acid  is  present ;  but,  as 
we  have  seen,  the  gastric  juice  is  further  capaVjle  of  effecting  the 
coagulation  of  lime-casein  even  though  hydrochloric  acid  is  absent. 
This  is  brought  about  through  the  specific  activity  of  the  milk- 
curdling  ferment  (chymosin) ;  but  it  is  to  be  noted  that  the  coagula- 
tion of  milk  is  in  this  case  not  directly  comparable  to  the  action  of 
an  acid ;  for  Avhile  the  latter  merely  brings  about  the  separation  of 
the  casein  by  the  removal  of  its  basic  component,  Hammarsten  has 
shown  that  the  chymosin  previously  causes  a  partial  decomposition 
of  the  lime-casein  by  hydrolysis.  As  a  result,  a  small  amount  of  an 
albumose-like  substance  is  split  off,  which  is  found  in  the  whey, 
while  the  greater  portion  of  the  lime-casein  is  transformed  into  so- 
called  lime-paracasein.  The  paracasein  is  likewise  a  nucleo-albumin 
with  acid  properties,  and  forms  salts  with  the  alkalies  and  lime, 
Avhich,  like  those  of  casein,  are  readily  soluble  in  water.  These 
salts  are  then  further  apt  to  combine  with  soluble  calcium  salts 
to  form  double  salts,  which  are  insoluble  in  nearly  neutral  solu- 
tions. As  the  milk  is  nearly  neutral  in  reaction,  and  as  soluljle 
calcium  salts  are  at  the  same  time  present,  coagulation  consequently 
occurs.  The  resulting  clot  constitutes  what  is  commonly  known  as 
cheese. 

In  the  absence  of  soluble  calcium  salts  coagulation  does  not 
occur  after  addition  of  the  chymosin.  Lime-paracasein,  however,  is 
manifestlv  formed,  as  upon  subsequent  treatment  with  a  soluble 
calcium  salt  the  fluid  coagulates  in  the  usual  manner.  That  the 
ferment  takes  no  part  in  the  process  of  coagulation  itself,  but  merely 
prepares  the  lime-casein,  as  it  were,  for  this  end,  can  readily  be 
demonstrated  by  boiling  the  solution  of  chymosin  and  lime-casein 
after  having  been  kept  at  a  temperature  of  about  35°  C.  for  a  few 
minutes.  The  ferment  is  thus  destroyed,  but  coagulation  occurs 
nevertheless  if  a  soluble  calcium  salt  is  now  added. 

The  pepsin  of  the  gastric  juice  plays  no  ])art  whatever  in  the 
coagulation  of  the  milk.  But  after  this  has  taken  place  the  actual 
digestion  of  the  precipitated  lime-paracasein  begins.  As  I  have 
pointed  out,  this  is  then  decomposed,  with  the  formation  of  a  para- 
nuclein  and  allnimin,  which  latter  is  digested  in  the  normal  manner, 
A  hetero-albumose,  however,  is  not  formed  during  the  process. 

From  the  above  considerations  it  is  clear  that  all  those  factors 
which  tend  to  increase  the  amount  of  soluble  lime  salts  in  the  milk 
will  increase  the  tendency  of  the  lime-casein  to  coagulate  upon  the 
subsequent  addition  of  chymosin,  while  this  is  diminished  if  the 
soluble  salts  are  transformed  into  the  insoluble  form  or  if  their 
amount  is  diminished.  It  is  for  this  reason  also  that  boiled  milk 
does  not  coagulate  so  rapidly  as  fresh  milk,  as  the  free  carbonic  acid, 
which  holds  a  certain  amount  of  calcium  in  solution,  is  thereby 
removed.     The  common  addition  of  lime-water  to  milk   similarly 


THE  MILK.  445 

increases  the  tendency  to  coagulation,  but  does  not  render  it  more 
digestible,  as  is  generally  supposed. 

The  coagulation  of  the  milk  which  occurs  spontaneously  on 
standing  is  analogous  to  that  which  residts  upon  the  addition  of  a 
mineral  acid,  and  is  referable  to  the  formation  of  lactic  acid  from 
lactose  as  a  result  of  bacterial  action.  The  phenomenon  has 
nothing  in  common  with  the  coagulation  which  results  from 
chymosin,  and  is  merely  the  outcome  of  the  withdrawal  of  the  lime 
salts  from  the  lime-casein  and  the  liljcration  of  the  latter. 

From  the  fact  that  the  coagulum  which  results  in  cows'  milk 
upon  the  addition  of  chymosin  is  much  tougher  and  denser  than 
that  which  is  obtained  with  human  milk,  it  has  been  concluded  that 
the  casein  of  the  two  is  not  identical.  Soxhlet,  however,  has  shown 
that  the  density  of  the  coagulum  is  primarily  dependent  upon  the 
concentration  of  the  casein  solution  and  the  amount  of  soluble  cal- 
cium salts  and  acid  phosphates  present.  As  this  is  much  greater  in 
cows'  milk  than  in  human  milk,  it  follows  that  marked  differences 
must  thus  exist.  There  is  evidence  to  show,  nevertheless,  that 
different  forms  of  casein  occur.  Elementary  analysis  of  human 
casein  (Hammarsten)  and  cows'  casein  ( Wroblewski)  has  given 
the  following  results : 

Human,  C,  52.96 ;   H,  7.05;   N,  15.65  ;   S,  0.75;    P,  0.84  ;   O,  22.78  per  cent. 
Cows',     C,  52.24;   H,  7.32;  N,  14.97  ;    S,  1.11;    P,  0.68 ;   0,23.66     "      " 

The  difference  is  here  especially  noticeable  in  the  amount  of  sul- 
phur. Human  casein,  moreover,  is  not  so  readily  ])recipitat€Kl  by 
salting  or  by  the  addition  of  acids,  and  does  not  always  coagulate 
with  chymosin.  The  gastric  juice,  it  is  true,  can  precipitate  the 
substance,  but  it  readily  dissolves  in  an  excess  without  leaving  any 
residue  of  nuclein.  From  this  observation  Szontagh  has  concluded 
that  human  casein  is  in  reality  no  nucleo-albumin.  But  aside  from 
these  data  we  have  abundant  evidence  that  human  casein  and  cow.s' 
casein  are  not  identical,  in  the  fiict  that  no  modification  of  cows' 
milk,  however  produced,  is  so  readily  digested  by  the  infant  as  is 
human  milk. 

Like  all  albumins,  casein  is  optically  active  ;  its  specific  rotation 
in  neutral  solution  is  —  80  deo;rees. 

The  isolation  of  the  casein  from  milk  will  be  described  below,  in 
association  with  the  isolation  of  the  soluble  albumins.  These,  as  I 
have  already  said,  are  lactalbumin  and  lactoglobulin. 

Lactalbumin. — Lactalbumin  is  found  both  in  human  milk  and 
cows'  milk,  and  is  manifestly  closely  related  to  the  common  serum- 
albumin  of  the  blood-plasma.  Its  specific  rotation,  however,  is 
markedly  less,  viz., — 37  degrees,  as  compared  with  — 62.6  to — 64.6 
degrees.  Its  composition  according  to  Sebelien  is  C,  52.19  per  cent. ; 
H,  7.18  ;  N.  15.77  ;  S,  1.73  ;  and  O,  23.13  ;  while  that  of  serum- 
albumin  is  given  as  C,  52.25-53.06  per  cent. ;  H,  6.65-6.85 ;  N, 
15.88-16.04;  S,   1.8-2.25;  and    O,    22.25-22.97  (Hammarsten). 


446  THE  GLANDULAR   ORGANS. 

Lactoglobulin. — The  lactoglobulin  which  has  been  isolated 
from  cows'  milk  seems  to  be  identical  with  the  serum-globulin 
of  the   blood.     It  requires  no  further  description. 

That  still  other  albuminous  substances  may  occur  in  the  milk  is 
possible ;  but  if  so,  they  are  present  only  in  traces  and  have  not  as 
yet  been  identified.  Albumoses  and  peptones  are  not  found  in 
fresh  milk.  According  to  Siegfried,  a  phosphor-carnic  acid  can  be 
isolated  from  milk  after  removal  of  the  casein  and  the  coagulable 
albumins  ;  this,  however,  is  supposedly  not  identical  with  that  found 
in  muscle-plasma. 

Origin  of  the  Albumins. — Casein,  as  has  been  stated,  is  a  specific 
product  of  the  activity  of  the  mammary  glands,  and  is  probably 
formed  from  the  complex  nucleo-glucoproteid  which  occurs  in  the 
functionally  active  organ.  As  this  is  not  found  in  the  milk,  we  may 
conclude  that  after  its  formation  it  is  decomposed  and  probably 
yields  casein,  on  the  one  hand ;  while  its  reducing  radicle  may  be 
concerned  in  the  production  of  lactose. 

Of  the  origin  of  lactoglobulin  and  lactalbumin,  nothing  is  known ; 
but,  as  I  have  said,  the  former  is  probably  identical  with  the  serum- 
globulin  of  the  blood,  while  in  the  case  of  the  latter  we  may 
imagine  that  it  has  originated  through  a  peculiar  transformation  of 
the  scrum-all)umiu. 

Isolation  of  the  Albumins  of  the  Milk. — Isolation  of  Casein. — 
The  milk  is  diluted  with  four  times  its  volume  of  water  and  acidi- 
fied with  acetic  acid  to  the  extent  of  0.75-1.0  pro  mille.  On  stand- 
ing, the  casein  separates  out  and  is  filtered  off.  It  is  purified  by 
repeated  solution  in  water  with  the  aid  of  a  little  caustic  alkali, 
filtration,  and  reprecipitation  with  acetic  acid.  It  is  then  washed 
with  water  and  freed  from  traces  of  fat  by  means  of  ether-alcohol. 
The  greater  portion  of  the  fat  remains  on  the  first  filter. 

Isolation  of  Lactoglobulin. — The  milk  is  saturated  with 
common  salt  in  substance,  which,  precipitates  the  lime-casein  to- 
gether with  a  small  portion  of  the  globulin.  If  then  the  neutral 
filtrate  is  saturated  with  magnesium  sulphate  at  30°  C.,the  remain- 
ing portion  of  the  lactoglobulin  is  obtained.  This  is  purified  as 
described  on  page  314. 

Isolation  of  Lactalbumin. — The  lime-casein  and  globulin  are 
first  precipitated  by  salting  with  magnesium  sul])hate  in  substance  at 
30°  C.,  and  are  filtered  off.  In  the  filtrate  the  lactalbumin  can 
then  be  demonstrated  by  acidifying  with  acetic  acid  to  the  extent 
of  a  little  less  than  1  per  cent,  and  boiling  or  by  salting  with  am- 
monium sulphate  or  sodium  sulphate  in  substance.  The  albumin 
is  filtered  off  aud  purified  as  described  on  page  339. 

Quantitative  Estimation  of  the  Total  Albumins. — To  this  end,  a 
few  grammes  of  milk  are  diluted  with  water,  treated  with  a  small 
amount  of  sodium  chloride  solution,  and  precipitated  with  tannic 
acid  or  phosphotungstic  acid  in  excess.  In  the  precipitate,  which  is 
washed  with  water,  the  amount  of  nitrogen  is  then  estimated  by 


THE  MILK.  447 

Kjeldahl's  method.  By  multiplying  the  result  by  6.37  in  the  case 
of  cows'  milk,  or  h\  6.34  with  human  milk,  the  corresponding 
amount  of  albumin  in  ascertained.  The  nitrogen  of  some  of  the 
extractives  is  included  in  the  result,  but  may  be  ignored.  In  cows' 
milk  it  represents  about  one-sixteenth  of  the  total  amount  of  nitro- 
gen, and  in  human  milk  about  one-eleventh. 

Separate  Estimation  of  the  Casein  and  the  Soluble  Albumins. — A 
few  grammes  of  milk  are  diluted  with  two  or  three  volumes  of  a 
saturated  solution  of  magnesium  sulphate,  and  are  then  saturated 
with  the  salt  in  substance.  In  the  precipitate,  which  is  washed  with 
a  saturated  solution  of  the  salt,  the  nitrogen  is  then  determined  as 
above.  The  result  multi[)lied  by  6,37  indicates  the  amount  of 
casein.  The  amount  of  lactalbumin  can  be  ascertained  by  deduct- 
ing the  value  found  for  casein  from  the  total  amount  of  albumin,  or 
by  diluting  the  filtrate,  after  separation  of  the  casein,  precipitating 
with  tannic  acid,  and  determining  the  amount  of  nitrogen  as  before. 
In  this  case  also  we  multiply  by  6.37. 

The  results  for  casein  thus  obtained  are  not  absolutely  accurate, 
as  the  globulin  is  likewise  precipitated  by  magnesium  sulphate. 
Its  amount,  h(iwever,  is  so  small   that  it  may  well  be  disregarded. 

The  Fats. — The  fats  which  are  found  in  the  milk,  viz.,  in  butter, 
are  essentially  the  same  as  those  which  occur  elsewhere  in  the 
animal  body,  viz.,  stearin,  palmitin,  and  olein.  In  addition,  how- 
ever, we  also  find  small  amounts  of  the  triglycerides  of  myristinic 
acid,  butyric  acid,  and  capronic  acid,  and  traces  of  caprylic  acid, 
caprinic  acid,  laurinic  acid,  and  arachinic  acid. 

Stearin,  palmitin,  and  olein  constitute  about  98  per  cent,  of  the 
total  amount,  and  of  these,  olein  represents  about  29.4-39.2  per 
cent.  As  a  consequence  of  the  large  quantity  of  olein  which  is 
thus  present,  the  meltintj-point  of  butter  is  relativelv  low,  viz., 
31°-34°  C,  while  it  solidifies  between  19°  and  24°  C.  ' 

In  addition  to  the  neutral  fits,  butter  also  contains  about  7  per 
cent,  of  volatile  fatty  acids,  of  which  3.7-5.1  per  cent,  are  repre- 
sented by  butyric  acid  and  2-3.3  per  cent,  by  capronic  acid. 

Formic  acid  has  been  found  in  butter  which  had  been  exposed  to 
sunlight. 

Of  the  origin  of  the  fats  which  are  found  in  milk  we  know  that 
they  are  to  a  large  extent  derived  from  albumins,  and  I  have  already 
pointed  out  that  their  amount  increases  with  a  diet  that  is  rich  in 
such  material,  even  though  no  fat  is  ingested  at  all.  They  diminish 
materially  if  fat  alone  is  ingested,  and  are  not  increased  if  much  fat 
is  administered,  while  the  ino-estion  of  albumin  remains  constant. 
They  are  probably  Ibrmed  in  the  gland  directly,  and  on  micro- 
scopical examination  it  is  possible  to  demonstrate  their  presence  in 
the  cells,  in  the  form  of  fine  globules,  which  are  soluble  in  ether, 
and  are  colored  black  on  treating  with  osmic  acid. 

To  isolate  the  individual  acids  which  enter  into  the  composition 


448  THE  GLANDULAR  ORGANS. 

of  the  neutral  fats,  tlie  butter  is  first  saponified,  when  the  resulting 
soaps  may  be  separated  from  each  other  according  to  the  usual 
methods  of  analysis. 

Quantitative  Estimation. — The  amount  of  fat  in  milk  is  most 
conveniently  estimated  densimetrically  with  Soxhlet's  apparatus. 
To  this  end,  a  known  amount  of  milk  is  mixed  with  a  solution 
of  sodium  hydrate  and  the  fat  extracted  with  a  definite  quantity 
of  ether.  The  ethereal  solution  is  allowed  to  separate,  and  is 
then  forced  into  a  glass  cylinder  provided  Avith  an  aerometer.  From 
the  specific  gravity  the  percentage  of  fat  is  then  read  off*  from  a  table 
which  accompanies  the  apparatus.  The  latter  is  so  constructed  that 
evaporation  of  the  ether  cannot  occur. 

In  the  absence  of  such  an  apparatus  the  amount  of  fat  can  be 
ascertained  gravimetrically  as  follows  :  20  c.c.  of  milk  are  treated 
with  a  small  amoimt  of  sodium  hydrate  solution,  and  are  extracted 
with  80  c.c.  of  ether  which  has  been  saturated  with  water.  This  is 
done  by  shaking  in  a  tightly  closed  bottle.  After  the  ethereal  ex- 
tract has  entirely  separated,  60  c.c.  are  placed  in  a  weighed  beaker  ; 
the  ether  is  allowed  to  evaporate  ;  the  residue  is  dried  and  weighed. 
The  result  is  calculated  out  for  80  c.c.  of  the  ethereal  extract, 
corresponding  to  20  c.c.  of  milk. 

Lactose. — In  the  animal  body  lactose  is  found  only  in  the  milk,  if 
we  disregard  the  small  amounts  that  may  appear  in  the  urine  of  nurs- 
ing females,  and  which  must  hence  of  necessity  occur  also  in  the 
blood.  It  is  formed  in  the  mammary  glands,  and  may  possibly 
be  related  to  the  reducing  suljstance  which  results  from  the  nucleo- 
glucoproteid  when  this  is  boiled  with  mineral  acids.  On  exposure 
to  the  air  it  undergoes  a  peculiar  fermentation,  with  the  formation 
of  lactic  acid.  This,  in  turn,  combines  with  the  calcium  of  the 
lime-casein,  and  as  a  result  the  casein  separates  out,  and  constitutes 
what  is  popularly  termed  clabber.  On  further  standing,  this  con- 
tracts, and  finally  floats  in  a  clear,  light-yellow  fluid — the  acid  vhey. 
The  fermentation  in  question  is  produced  by  definite  micro-organ- 
isms, of  which  fourteen  varieties  are  now  known. 

On  inversion,  lactose  is  decomposed  into  glucose  and  galactose 
(see  page  72). 

Isolation. — To  isolate  lactose  from  milk,  this  is  first  curdled  by 
the  addition  of  chymosin.  The  filtrate  is  slightly  acidified  with 
acetic  acid  and  boiled,  so  as  to  remove  the  coagulable  albumins.  The 
second  filtrate  is  then  concentrated  to  a  small  volume,  when  on  cool- 
ing the  lactose  crystallizes  out.  To  purify  the  substance,  this  is  dis- 
solved in  water,  decolorized  with  animal  charcoal,  and  recrystallized 
by  evaporation.  It  is  thus  obtained  in  the  form  of  white  rhombic 
prisms,  which  are  soluble  in  water,  but  insoluble  in  absolute  alcohol. 
The  substance  has  a  somewhat  sweetish  taste,  and  contains  one  mole- 
cule of  water  of  crystallization,  which  rapidly  escapes  at  130°  C. 

Estimation. — To  estimate  the  amount   of  lactose,  the  milk  must 


THE  MILK.  449 

first  be  freed  from  fats  and  albumins.  To  this  end,  it  is  most  con- 
venient to  dilate  with  water  and  to  remove  the  casein  bv  the  cau- 
tious addition  of  acetic  acid.  The  resulting  precipitate,  which 
contains  both  the  casein  and  the  fat,  is  filtered  off  and  the  filtrate 
boiled.  After  tlie  removal  of  the  precipitated  coagulablo  albumins, 
the  sugar  is  then  estimated  in  the  filtrate  by  titrating  with  Knapp's 
solution,  as  described  in  the  section  on  the  Urine.  Ten  c.c.  of  the 
reagent  correspond  to  0.031  gramme  of  lactose,  providing  that  the 
solution  contains  from  0.5  to  1  per  cent,  of  sugar. 

In  addition  to  lactose,  the  milk  contains  also  small  amounts  of  a 
reducing  substance,  which  is  sup])osedly  identical  with  Landwehr's 
animal  gum.  It  is  possible,  however,  that,  as  in  the  case  of  the 
reducing  substances  of  the  mucins  and  mucoids,  chondroitin-sul- 
phuric  acid  or  an  allied  substance  may  be  responsible  for  the 
reactions. 

Extractives. — Among  the  extractives  of  the  milk,  which  com- 
prise traces  of  urea,  kreatin,  kreatinin,  xanthin-bases,  lecithins, 
cholesterin,  and  citric  acid,  the  latter  is  of  especial  interest,  as  it 
is  apparently  also  formed  in  the  mammary  glands,  and  is  not  refer- 
able to  the  ingestion  of  the  substance  as  such.  It  has  been  found  in 
human  milk  as  well  as  in  cows'  milk,  and  is  notably  present  in  com- 
bination with  calcium.  Its  amount  in  cows'  milk  is  given  as  0.25 
per  cent.  Human  milk  contains  a  somewhat  smaller  amount  of 
citric  acid  ;  it  varies  between  0.024  and  0.07  per  cent.  (Sieber) ;  the 
higher  values  are  found  between  the  sixth  and  the  eleventh  month 
of  lactation. 

To  the  presence  of  citric  acid  apparently  the  reaction  of  Umikoff 
is  due.  To  make  the  test,  about  5  c.c.  of  milk  are  treated  with  one- 
half  the  amount  of  a  10  per  cent,  solution  of  ammonium  hydrate  and 
heated  on  a  water- bath  at  60°  C  for  fifteen  to  twenty  minutes. 
Human  milk  then  assumes  a  violet-reddish  color,  which  is  the  more 
intense  the  older  the  milk  in  reference  to  the  time  of  lactation.  Cows' 
milk  treated  in  the  same  manner  assumes  a  yellow,  at  most  a  yellowish- 
brown  color,  so  that  it  is  thus  possible  to  distinguish  human  milk 
from  cows'  milk.  According  to  Sieber,  it  is  also  possible  thus  to 
distinguish  the  milk  of  the  earlier  months  of  lactation  from  that  of 
the  later  months ;  after  the  eighth  month,  however,  the  reaction  no 
longer  gives  uniform  results,  but  is  sometimes  intense,  and  at  others 
quite  feeble.  Sieber  has  ascertained  that  the  difference  between 
human  and  cows'  milk  in  this  respect  is  probably  primarily  depen- 
dent upon  the  differing  amount  of  calcium  salt  that  is  present.  On 
heating  cows'  milk  with  ammonia  all  the  citric  acid  is  precipitated 
as  calcium  citrate,  while  in  the  case  of  human  milk,  which  contains 
only  one-sixth  the  amount  of  calcium,  a  certain  proportion  of  the 
citric  acid  remains  in  solution. 

The  formula  of  the  acid  is  CH,.COOH.CYOH).COOH.CH2.- 
COOH,  viz.,  CgHgO- ;  it  is  thus  oxy-tricarballylic  acid. 

29 


450  THE  GLANDULAR   ORGANS. 

Ferments. — At  least  three  ferments  seem  to  occur  in  cows'  milk^ 
viz.,  a  railk-trypsin,  a  milk-katalase,  and  a  milk-peroxydase.  In 
addition  Babcock  and  Russell  have  described  a  galaktase.  In 
human  milk  there  is  a  diastase  (absent  in  cows'  milk),  very  little 
if  any  peroxydase,  but  more  katalase  than  in  cow^'  milk  ;  and  in 
addition  a  proteolytic  ferment.  Possibly  still  other  ferments  are 
present. 

Colostrum. 

The  term  colostrum  is  applied  to  the  secretion  of  the  mammary 
glands  which  is  furnished  by  the  female  animal  during  the  first 
days  of  lactation,  and  which  may  also  be  expressed  from  the  glands 
during  a  variable  period  jireceding  parturition. 

On  microscopical  examination  such  fluid  is  seen  to  contain  innum- 
erable fat-globules,  and  in  addition  a  variable  number  of  granular 
cells,  which  are  capable  of  manifesting  amoeboid  movements.  These 
are  termed  colostrum-corpuscles,  and  are  commonly  regarded  as 
leucocytes.  This,  however,  is  doubtful.  According  to  Woodward, 
they  have  a  small  irregular,  but  much  degenerated  nucleus.  Of  the 
granules,  a  few  are  stained  by  osmic  acid,  while  none  of  them  takes 
up  either  acid,  neutral,  or  basic  dyes.  In  their  reactions  they  show 
the  characteristics  of  proteid  material. 

The  secretion  is  a  thick  yellowish  fluid,  of  an  alkaline  and  some- 
times acid  reaction,  and  a  specific  gravity  that  is  much  higher  than 
that  of  true  milk.  In  the  cow  this  varies  between  1.046  and 
1.080,  and  in  the  human  female  between  1.040  and  1.060.  This  is 
principally  owing  to  the  presence  of  large  amounts  of  lactalbumin 
and  lactoglobulin.  As  a  consequence,  the  colostrum  coagulates 
on  boiling,  while  true  milk,  as  we  have  seen,  is  then  covered  merely 
by  a  skin,  which  is  composed  of  casein  and  calcium  phosphates.  The 
total  quantity  of  the  coagulable  albumins  may  reach  15  per  cent,, 
while  in  milk  about  0.5  per  cent,  is  the  rule. 

The  amount  of  casein  and  of  mineral  salts  in  colostrum  is  also 
somewhat  greater  than  in  milk,  and  it  is  further  stated  that  more 
lecithin  and  cholesterin  is  present.  The  quantity  of  fat  is  practi- 
cally the  same,  while  that  of  lactose  is  somewhat  smaller.  As  a 
result  of  the  increase  in  the  amount  of  albumins  and  of  mineral  salts, 
the  total  solids  are  also  proportionately  increased,  and  may  amount 
to  25.3  per  cent,  in  cows'  colostrum,  as  compared  with  12.8  per  cent, 
in  the  case  of  the  milk. 

The  quantitative  composition  of  the  colostrum  after  parturition  is 
rapidly  altered,  so  that  after  a  few  days  already  the  normal  compo- 
sition of  true  milk  is  approached.  This  is  well  shown  in  the  follow- 
ing table,  which  is  taken  from  Gautier.  The  results  have  reference 
to  the  human  being  and  the  cow,  and  are  expressed  in -percentages  : 


THE  REPRODUCTIVE  GLANDS.  451 

Human  Beinc4. 

Nine  days  be-    Day  of  partu-    Twenty-four  Nine  days  later, 
fore    partu-       rition.  hours  after 

rition.  parturition. 

Water 85.86  82.80  84.30  88.580 

Solids 14.15  17.20  15.70  .  11.420 

Albumins      ....      8.071  ^^^  _    _  3^90 

Lasein J 

Fat 2.35  5.00  .    .  3.530 

Lactose      3.63  7.00  .    .  4.300 

Salts  and  extractives     0.54  .    .  0.512  0.169 

Cow. 

Immediately        Twenty-four     Three    days       Average  of 
after  partu-  hours  later.  later.  30  analyses, 

rition. 

Water 73.07  82.38  78.70  74.05 

Solids    . 26.93  17.62  21.30  25.95 

Albumins      ....  16.56  4.50  7.50  13.62 

Casein 2.65  4.50  7.30  4.66 

Fat 3.54  4.75  4.00  3.43 

Lactose 3.00  2.85  1.50  2.66 

Salts  and  extractives  1.18  1.02  1.00  1.58 

I  also  append  a  few  analyses  of  cows'  colostrum,  which  I  have 
taken  from  G.  Simon  (the  analysis  were  made  on  successive  dogs) : 

Specific  gravity  .    .    .    1.0705         1.0690  1.03G6  1.0359  per  cent. 

Fat 3.00            3.00  3.03  3.54  " 

Solids 23.85  23.60  13.30  13.20  " 

Total  albumins  ...  17.00  16.87  4.93  4.35 

Casein 5.50            4.47  3.02  3.06  " 

Albumin 11.93  11.79  1.91  1.15  " 

Extractives 0.07  0.07  0.04  " 

As  I  have  already  indicated,  probably  all  colostra  color  active  tinc- 
ture of  guaiacum  blue  even  in  the  absence  of  hydrogen  peroxide,  and 
also  react  with  the  Rohmann-Spitzer  mixture.  The  reaction  is  due 
to  a  globulin-oxydase. 

So-called  witchh  milk  is  the  fluid  which  can  be  expressed  from 
the  mammary  glands  of  both  sexes  immediately  after  birth.  Its 
qualitative  composition  is  the  same  as  that  of  milk.  Like  the  colos- 
trum, it  is  said  to  contain  colostrum-corpuscles.  According  to 
Schlossberger,  HaufP,  and  others,  it  contains  from  1.05  to  2.8  per 
cent,  of  albumin,  0.82  to  1.46  per  cent,  of  fat,  and  0.9  to  6  per 
cent,  of  lactose.  It  thus  contains  a  smaller  amount  of  water  than 
the  milk.      The  secretion  ceases  several  weeks  after  birth. 

Uterine  milk  is  a  fluid  which  can  be  obtained  from  the  uterine 
glands  of  ruminants  after  careful  separation  of  the  chorion  villi.  It 
has  the  appearance  of  cream,  and  is  morphologically  and  chemically 
quite  similar  to  colostrum. 

THE   REPRODUCTIVE   GLANDS. 

The  Testicles. — Of  the  chemical  composition  of  the  testicles  as 
such,  little  is  known.  Aside  from  the  albuminoids  which  enter  into  the 
construction  of  the  supporting  tissues  of  the  glands,  and  the  extrac- 


452  THE  GLANDULAR   ORGANS. 

tives  Avliicli  are  common  to  all  organs  of  the  body,  we  notably  meet 
with  albumins,  among  which  the  nucleins  are  especially  abundant. 
In  addition,  serum-albumin,  a  globulin,  and  a  substance  which 
apparently  belongs  to  the  hyalins,  have  been  encountered.  Of  mineral 
salts,  we  notably  meet  with  the  chlorides  of  sodium  and  potassium. 
The  reaction  of  the  glands  is  alkaline. 

The  Semen. — The  specific  product  of  the  functional  activity  of 
the  testicles  is  represented  by  the  semen,  and  notably  its  morpho- 
logical elements,  the  spermatozoa,  which  result  from  the  spermato- 
genetic  cells  through  a  complicated  process  of  metamorphosis,  in 
which  the  cell-nuclei  are  especially  concerned.  On  its  passage  to 
the  outside  the  testicular  fluid  is  mixed  with  the  secretions  of  the 
seminal  vesicles,  the  glands  of  Cowper,  and  notably  with  the  secre- 
tion of  the  prostate  gland. 

Recently  ejaculated  semen  is  a  markedly  viscid,  white  or  yellow- 
ish-white, opaque  fluid  of  the  appearance  of  milk,  in  which  micro- 
scopical examination  reveals  the  presence  of  innumerable  sperma- 
tozoa, and  a  few  hyalin  globules,  which  are  derived  from  the  seminal 
vesicles ;  further,  isolated  testicular  and  urethral  cells,  prostatic 
corpuscles,  and  cellular  bodies  enclosing  lecithin  granules,  besides  a 
large  number  of  free  granules,  which  are  apparently  of  an  albumin- 
ous nature.  In  a  fresh  specimen  the  normal  spermatozoa  are  ac- 
tively motile,  and  continue  so  for  a  variable  length  of  time  if  evapo- 
ration is  prev^euted.  The  movements  are  in  all  likcbhood  analogous 
to  those  of  the  cilia  of  certain  epithelial  elements  of  the  1)ody,  and  are 
arrested  by  the  addition  of  water,  dilute  acids,  alcohol,  ether,  strongly 
alkaline  solutions,  etc.  In  dilute  alkaline  solutions,  on  the  other 
hand,  and  those  of  the  neutral  salts  they  continue  for  a  long  time. 

Semen  is  heavier  than  water,  and  falls  to  the  bottom  as  a  jelly- 
like mass  :  at  the  same  time  a  light  flocculent  precipitate  develops, 
which  consists  of  the  so-called  fibrin  of  Henle.  On  exposure  to  the 
air  it  is  apparently  coagulated,  but  later  becomes  liquid,  as  before. 
Its  reaction  is  neutral  or  slightly  alkaline  ;  the  alkalinity  corresponds 
to  about  0.148  per  cent,  of  sodium  hydrate.  The  specific  gravity 
varies  from  1.020  to  1.039. 

Testicular  semen,  in  contradistinction  to  that  which  has  been 
ejaculated,  is  said  to  be  odorless.  After  emission,  however,  an  odor 
develops  which  is  suggestive  of  glutin,  and  is  supposedly  referable 
to  the  presence  of  an  alkaloidal  substance — sjienniiu  In  combina- 
tion with  phosphoric  acid,  this  is  found  in  the  secretion  of  the 
prostate  gland,  as  phosphate  of  spermin,  and  is  partly  decomposed 
on  exposure  to  the  air,  with  liberation  of  the  free  base  (see  below). 

A  quantitative  analysis  of  human  semen  gave  the  following 
results  (Slowtzoif ) ;  the  figures  have  reference  to  100  parts  of  the 
fresh  material : 


THE  REPRODUCTIVE  GLANDS.  453 

Water 90.321 

Solids 8.679 

Salts  ...••. 0.901 

Organic  material 8.778 

Extractives 6.278 

Albuminous  material  and  nucleins 2.092 

The  Spermatic  Liquid. — The  liquid  in  which  the  spermatozoa 
are  sitspended  is  nearly  tran.sparent.  It  contains  a  small  amount  of 
mucin  (?) ;  a  nucleo-albumin,  which  has  been  termed  spermatin,  and 
which  is  precipitated  by  acetic  acid,  but  is  readily  soluble  in  an 
excess  of  the  reagent ;  common  albumin  ;  an  albumose-like  body 
which  can  be  precipitated  on  two-thirds  saturation  with  ammonium 
sulphate ;  cerebrin  and  lecithins,  phosphate  of  spermin,  and  various 
mineral  salts,  among  which  sodium  chloride  and  the  phosphates  of  the 
alkaline  earths  predominate. 

Spermin. — Spermin,  as  stated  above,  occurs  in  the  spermatic  liquid 
in  combination  with  phosphoric  acid,  as  phosphate  of  spermin  ;  it 
is  viewed  as  ethylenimin,  C^H^N,  and  is  manifestly  closely  related  to 
the  diethylene  diamin  (piperazin)  of  Ladenburg  and  Abel. 

To  the  free  base  the  peculiar  odor  of  the  semen  is,  as  I  have  said, 
supposedly  due.  This  disappears  after  a  short  wliile,  owing  to 
a  polymerization  of  the  ethylenimin  to  diethylene  diamin  (piperazin) 
as  shown  in  the  equation  : 

XCH^  \NH/ 

The  phosphate  can  be  readily  obtained  in  crystalline  form  on  slow 
evaporation  of  the  semen,  but  may  also  separate  out  spontaneously 
on  standing  for  about  twenty-four  hours.  It  occurs  in  the  form  of 
hexagonal  pyramids,  which  appear  under  the  microscope  as  flat 
needles.  They  are  soluble  in  dilute  acids  and  alkalies,  as  also  in 
ammonia,  less  readily  so  in  hot  water,  and  are  insoluble  in  alcohol, 
ether,  and  chloroform.  These  crystals  are  known  as  Bottcher's 
spermin-crystals,  and  are  probably  identical  with  the  so-called 
Charcot-Leyden  crystals,  which  are  commonly  found  in  asthmatic 
sputa,  and  also  occur  in  the  blood  and  lymph-gland  of  leuksemic 
patients.  They  have  likewise  been  observed  in  dried  egg-albumin 
and  in  anatomical  specimens  preserved  in  alcohol,  and  also  develop 
in  red  bone-marrow  that  has  been  exposed  to  the  air  for  a  few  days. 
Heated  to  100°  C,  the  crystals  turn  yellow  and  melt  near  170°  C, 
but  are  at  the  same  time  decomposed. 

To  isolate  the  free  base,  the  semen  is  extracted  with  alcohol  and 
then  with  dilute  sulphuric  acid.  If  the  acid  extract  is  then  treated 
with  baryta-water  and  evaporated  at  a  low  temperature,  the  free 
base  is  obtained.  It  can  be  precipitated  from  its  solution  by  treating 
with  auric  chloride,  platinum  chloride,  argentic  nitrate,  tannic  acid, 
phosphotungstic  acid,  etc. 

Spermin    has    attracted    much    attention    of  late,    owing   to    the 


454  THE  GLANDULAR   ORGANS. 

stimulating  effect  which  the  substance  is  supposed  to  exert  upon  the 
oxidation  processes  of  the  body,  the  functions  of  tlie  central  nervous 
system,  and  the  reproductive  organs.  It  represents  the  active  prin- 
ciple of  Brown-Sequard's  elixir. 

The  Spermatozoa. — Our  knowledge  of  the  chemical  composi- 
tion of  the  spermatozoa  has  been  greatly  extended  within  recent 
years  through  the  researches  of  Kossel  and  his  pupils,  preceded  by 
those  of  Miescher  and  Piccard.  These  observers  were  able  to  show 
that  in  certain  fishes,  such  as  the  salmon,  sturgeon,  pike,  shad, 
herring,  and  mackerel,  substances  can  be  isolated  from  the  mature 
spermatozoa,  wdiich  apparently  represent  the  simplest  forms  of 
albumin,  and  are  now  collectively  termed  'protamins.  Their  general 
characteristics  have  already  been  described  (page  54).  These  pro- 
tamins, of  which  several  varieties  are  known,  and  which  yield  the 
hexon-bases  on  hydrolytic  decomposition,  are  supposedly  combined 
with  nucleinic  acids  to  form  nucleoproteids.  The  individual  nucle- 
inic  bases  which  enter  into  the  construction  of  the  nucleinic  acids 
are  the  common  forms,  which  are  also  found  elsewhere  in  the 
animal  body.  But  it  appears  that  the  spermatozoa  of  different 
animals  do  not  contain  all  forms.  In  the  case  of  the  salmon, 
Miescher  and  Piccard  thus  found  guauin  and  hypoxanthin,  while 
from  the  semen  of  the  carp  Kossel  obtained  adenin  and  hypoxanthin, 
as  also  small  amounts  of  xanthin,  but  no  guanin.  Inoko,  on  the 
other  hand,  claims  to  have  found  all  forms  in  the  semen  of  the 
salmon,  boar,  and  ox,  but  states  that  the  relative  amounts  of  the 
individual  forms  are  not  constant.  Immature  spermatozoa  appar- 
ently contain  histons  in  the  place  of  protamins. 

Analysis  of  the  spermatozoa  of  the  salmon  (Miescher) : 

Per  cent. 

Nucleins^ 48.68 

Protamins  (salmin) 26.76 

Other  albumins 10.32 

Lecithins      7.47 

Cholesterin       2.24 

Fats 4.53 

The  albumins  referred  to  in  this  talkie  have  not  been  studied  in 
detail.  One  of  them,  according  to  Miescher,  contains  4  jjer  cent,  of 
sulphur.  In  addition,  the  spermatozoa  are  said  to  contain  a  cere- 
broside,  which  is  similar  to  cerelDrin ;  also  a  very  considerable 
proportion  of  inorganic  salts,  which  are  essentially  represented  by 
phosphates. 

Detailed  analyses  of  the  spermatozoa  of  the  higher  animals  and  of 
man  are  not  yet  available. 

As  regards  the  composition  of  the  separate  parts  of  the  sperma- 
tozoa very  little  is  known,  but  it  seems  that  the  protamins,  com- 
bined with  nucleinic  acids,  are  the  mo.st  important  components  of 
the  head.     The  tails   are  dissolved   in  gastric  juice  on  prolonged 

*  The  nuclcins,  according  to  Kossel,  are  nucleinic  acids. 


THE  REPRODUCTIVE  GLANDS.  455 

digestion,  and  hence  probably  consist  of  albumins.  As  a  whole 
the  spermatozoa  are  exceedingly  resistant  to  ordinary  solvents.  They 
are  soluble  in  boiling  solutions  of  the  caustic  alkalies,  while  in 
concentrated  sulphuric  acid,  nitric  acid,  acetic  acid,  and  boiling- 
solutions  of  sodium  carbonate  they  dissolve  only  in  part.  They 
are  likewise  resistant  to  putrefactive  changes,  and  can  be  obtained 
from  dried  semen,  with  the  preservation  of  their  natural  form, 
by  placing  the  material  in  a  1  per  cent,  solution  of  sodium  chloride. 

The  Ovaries. — Thus  far  a  study  of  the  chemical  composition 
of  the  ovaries  has  not  revealed  any  special  points  of  interest. 
In  addition  to  collagen  and  mucins,  which  enter  into  the  con- 
struction of  the  su])porting  tissue  of  the  organs,  nucleins  and  true 
albumins  have  also  been  found,  and  are  probably  derived  from  the 
contained  ova  and  other  cellular  elements. 

The  most  important  constituents  of  the  cortex  of  the  gland,  viz., 
the  Graafian  follicles,  which  enclose  the  specific  product  of  the  func- 
tional activity  of  the  ovaries,  viz.,  the  ova,  have  for  obvious  reasons 
not  been  open  to  a  detailed  investigation.  The  contained  fluid 
is  apparently  serous  in  character.  After  the  discharge  of  the 
ova  the  remaining  follicles  are  first  filled  with  blood  from  the  torn 
vessels  of  the  vesicle,  and  are  subsequently  transformed  into  the 
so-called  corpora  lutea.  The  yellow  color  of  these  is  owing  to  lipo- 
chromes,  or  luteins,  of  which  an  amorphous  and  a  crystalline  form 
may  be  isolated  (see  also  page  462). 

The  Ovum. — Of  the  chemical  composition  of  the  ova  of  the 
human  being  and  mammals  in  general,  nothing  definite  is  known, 
as  it  is  impossible  to  collect  them  for  purposes  of  analysis.  The 
eggs  of  fishes,  amphibia,  reptiles,  and  especially  of  birds,  on  the 
other  hand,  can  readily  be  obtained  and  have  been  studied  in  greater 
detail.  In  the  following  pages  we  shall  confine  our  attention  to 
the  composition  of  birds'  eggs,  which  is  best  understood.  The  egg 
proper  is  here  surrounded  by  the  so-called  white  of  egg,  which  in 
turn  is  enclosed  in  a  double  membrane,  and  is  covered  by  the  shell. 
These  additional  structures,  however,  are  not  formed  in  the  ovary, 
but  are  produced  during  the  passage  of  the  egg  through  the  oviduct 
from  material,  which  is  here  secreted  by  the  lining  cells. 

The  Shell. — The  shell  consists  essentially  of  an  organic  matrix  of 
the  character  of  keratin,  which  is  largely  impregnated  with  lime 
salts.  Of  these,  calcium  carbonate  is  the  most  abundant,  and  con- 
stitutes about  90  per  cent,  of  the  weight  of  the  entire  shell.  In 
addition,  we  find  a  small  amount  of  magnesium  carbonate,  as  also 
phosphates  of  both  elements.  Water  is  present  to  the  extent  of 
only  about  1  per  cent.  The  pigments  met  with  in  birds'  eggs 
are  closely  related  to  the  Inliary  pigments,  and,  like  these,  are 
derived  from  the  common  pigment  of  blood.  The  odrhode'in,  which 
presents  a  reddish  or  brownish-red  color,  is  supposedly  identical 
with  haematoporphyrin  ;  while  the  blue  or  green  pigment,  which  is 


456  THE  GLANDULAR   ORGANS. 

termed  oocyanin,  is  composed  partly  of  biliverdin,  and  is  in  part  a 
blue  derivative  of  bilirubin. 

The  membranes  of  birds'  eggs  consist  essentially  of  keratin,  but 
contain  also  a  small  amount  of  mineral  salts,  of  which  calcium 
phosphate  is  the  most  abundant. 

In  fishes  and  amphibia  the  egg  envelope  is  represented  by  a  trans- 
parent gelatinous  material,  which  seems  to  consist  almost  exclusively 
of  mucin.  In  the  invertebrates  chitin  and  skeletins  take  the  place 
of  the  keratin  of  birds'  eggs,  but  in  some  the  latter  also  is  found. 

Tlie  weio-ht  of  the  shell  and  membranes  in  the  case  of  hens'  eggs 
represents  about  9  to  11  per  cent,  of  the  total  weight  of  the  egg, 
w^hile  the  albumen  constitutes  about  60.5  per  cent,  and  the  yolk, 
viz.,  the  ovum  proper,  the  remaining  29  per  cent.  The  total  weight 
of  hens'  eggs  may  vary  between  40  and  70  grammes. 

The  Albumen. — The  albumen  or  white  of  egg,  as  obtained  directly 
from  the  raw  egg,  appears  as  a  faintly  yellow,  exceedingly  viscid, 
semiliquid  matei'ial.  On  microscopical  examination  this  can  be 
shown  to  consist  of  compartments,  which  are  limited  by  very 
delicate  membranes,  and  enclose  the  albumen  proper.  These 
membranes  are  continuous  with  the  so-called  chalazse  and  the 
membranes  immediately  beneath  the  shell,  and  are,  like  these, 
composed  of  keratin. 

The  albumen  proper  may  be  separated  from  its  membranous  con- 
stituents by  pressing  the  material  through  a  cloth,  and  then  appears 
as  an  opalescent  fluid,  which  is  only  slightly  viscid,  and  can  be 
filtered  without  much  difficulty.  Its  reaction  is  distinctly  alkaline, 
and  the  specific  gravity  about  1,045.  On  boiling,  it  coagulates  to  a 
compact  mass,  which  in  the  case  of  hens'  eggs  is  entirely  opaque. 
In  some  birds,  however,  such  as  the  swallow,  the  crow,  the  finch, 
etc. — I.  e.,  in  true  nesting  birds — the  albumen  remains  transparent, 
owing  to  the  formation  of  alkaline  albuminates.  Such  albumen  has 
been  termed  tata-albumen.  It  may  be  produced  artificially  by  placing 
hens'  eggs  in  a  10  per  cent,  solution  of  sodium  hydrate  for  two  or 
three  days,  when  a  gradual  diffusion  of  alkali  occurs  into  the  albu- 
men.    On  subsequent  boiling,  this  appears  like  true  tata-albumen. 

Analysis  of  the  albumen  of  hens'  eggs  has  given  the  following 
results  : 

Per  cent. 

Water ■ 80.00-86.68 

Solids 13.32-20.00 

Albumins 11.50-12.27 

Extractives 0.38-  0.77 

Glucose 0.10-  0.50 

Fats  and  soaps ' traces 

Mineral  salts 0.30-  0.66 

Lecithins  and  cliolesterin traces 

According  to  Poleck  and  Weber,  the  mineral  ash  has  the  follow- 
ing composition,  calculated  for  100  parts: 


THE  REPRODUCTIVE  GLANDS.  457 

Sodium  (NaP) 23.56-32.93 

Potassium  fK,0) 27.66-28.45 

Calcium  (CaO) 1.74-  2.90 

Magnesium  (MgO) 1.60-  3.17 

Iron  (FeA) 0.44-  0.55 

Chlorine  (CI) 23.84-28.56 

Phosphoric  acid  (P^Os) 3.16-  4.83 

Carbonic  acid  (CO./) 9.67-11.60 

Sulphuric  acid  (Si)^)      1.32-  2.63 

Silicic  acid  (SiO,,) 0.28-  0.49 

Fluorine  (Fl) traces 

Of  these  constituents,  the  large  amount  of  sodium  chloride  is  espe- 
cially noteworthy,  and  shows  in  itself  that  the  albumen  is  in  reality 
a  secretory  product,  and  does  not  represent  a  mere  transudation  from 
the  blood-plasma. 

One  portion  of  the  bases  is  in  combination  with  the  albumins  of 
the  albumen,  while  the  remainder  exists  in  the  form  of  sulphates, 
phosphates,  and  notably  carbonates. 

The  slightly  yellow  color  of  albumen  is  referable  to  the  presence 
of  a  lipochrome,  which  can  be  demonstrated  on  spectroscopic  exam- 
ination. 

The  Albumins. — According  to  Gautier  and  some  of  the  older 
observers,  white  of  egg  (albumen)  contains  a  number  of  different 
albumins,  wliich  in  part  seem  to  belong  to  the  true  albumins  and  in 
part  to  the  globulins.  They  have  been  designated  as  a-,  /?-,  and 
/-ovalbumin,  and  a-  and  /9-ovoglobulin.  In  so  far  as  the  globulin 
fraction  is  concerned,  Langstein  has  recently  shown  that  it  consists 
of  two  portions,  one  of  which  after  precipitation  with  ammonium 
sulphate  is  insoluble  in  dilute  saline  solution,  Avhile  the  other  dis- 
solves. This  latter  fraction  was  further  subdivided  by  salting  with 
potassium  acetate  into  an  euglobulin  and  a  second  fraction  which  can 
subsequently  no  longer  be  precipitated  by  half-saturation  with 
ammonium  sulphate.  The  euglobulin  represents  about  two-thirds 
of  the  total  globulin  fraction. 

The  greater  portion  of  the  white  of  egg-  does  not  belong  to  the 
globulins,  and  can  be  obtained  in  crystalline  form,  viz.,  the  oval- 
bumin proper.  In  addition  we  find  a  fraction  which  is  not  crystal- 
lizable — the  so-called  conalbumin  of  Osborne  and  Campbell,  and 
finally  the  ovomucoid  of  Morner. 

Globulin  Fraction. — The  euglobulin  above  mentioned  gives  the 
biuret  reaction,  the  xanthoproteic  reaction,  that  of  Millon,  Adam- 
kiewicz,  and  markedly  also  that  of  Molisch.  It  is  precipitated,  on 
dialysis,  by  a  current  of  carbon  dioxide,  and  on  careful  addition  of 
dilute  acetic  acid.  It  is  soluble  in  an  excess  of  acids  and  in  dilute 
saline  solution,  but  readily  passes  over  into  an  insoluble  modification. 
In  a  2  per  cent,  solution  it  coagulates  between  64°  and  67°  C. 
Elementary  analysis  gave  the  following  results  :  C  ^  49.88  ;  H  = 
7.09  ;  N  =  14.36  ;  S  =  1.73  ;  O  =  26.92  (Langstein).  On  hydrol- 
ysis with  dilute  hydrochloric  acid  it  yields  11  per  cent,  of  gluco- 
samin. 


458  THE  GLANDULAR   ORGANS. 

Ovalbumin. — The  amount  of  crystallizable  ovalbumin  Mliich  can 
be  obtained  from  white  of  egg  varies  between  30  and  40  grammes 
pro  liter.  The  limits  of  precipitation  of  the  substance,  when 
purified  carefully  and  brought  into  a  10  per  cent,  solution,  containing 
the  normal  amount  of  alkali  of  white  of  egg,  viz.,  0.225  gramme 
of  sodium  carbonate  for  100  grammes  of  albumin,  were  62  and  68. 
Elementary  analvsis  of  a  carefully  purified  pre])aration  gave  the 
following  results  (Langstein)  :  C  =  52.46  ;  H  =  7.19  j  N  =  15.29  ; 
S^  1.34;  and  O  =  23.72.  On  hydrolysis  wnth  baryta  ovalbumin 
yields  a  polymeric  nitrogenous  carbohydrate,  which  S.  Friinkel  has 
termed  albamln,  and  which  appears  to  be  an  acetylated  glucosamin. 
Seemann  and  Langstein  could  both  demonstrate  the  formation  of 
glucosamin  on  hydrolysis  wdth  dilute  acids.  The  formula  which 
Friinkel  suggests  for  his  albamin  is  2(CcHgO^NH2)  +  HgO. 

Conalbumin. — Whether  the  conalbumin  is  in  reality  a  unity  and 
not  a  mixture  of  two  or  more  bodies  is  uncertain.  Elementary 
analysis  has  given  the  following  results  (Osborne  and  Campbell) : 
C  =  52.25;  H  =  6.99;  N  =  16.11;  S=1.7;  O  =  22.95.  Ac- 
cording to  Langstein,  the  substance  is  free  from  phosphorus.  Like 
all  the  other  albumins  of  white  of  egg,  the  conalbumin  also  yields 
glucosamin  on  hydrolytic  decomposition.  It  appears,  as  Hofmeister 
suggests,  that  the  glucosamin  in  the  white  of  egg  fulfils  the  same 
object  as  the  milk-sugar  in  the  case  of  milk. 

Analysis  of  the  Albumins  of  White  of  Eg-g. — The  whites  of 
a  large  number  of  eggs  are  freed  from  their  membranes  by  beat- 
ing; the  material  is  filtered  and  the  filtrate  treated  with  an  equal 
volume  of  an  accurately  neutral  solution  of  ammonium  sulphate. 
(Care  should  be  had  that  only  such  eggs  are  used  which  present  an 
alkaline  reaction  with  litmus.)  After  standing  for  one-half  hour 
the  precipitated  globulins  are  filtered  off.  The  clear  filtrate,  which 
usually  presents  a  reddish  color,  is  now  treated  wdth  one-fifth 
normal  solution  of  sulphuric  acid  until  the  fluid  becomes  opaque. 
If  any  crystals  of  ovalbumin  are  available,  a  few  are  added  to 
hasten  crystallization.  This,  however,  is  not  necessary.  The  solu- 
tion is  allowed  to  stand,  when  gradually  a  separation  of  crystals  of 
ovalbumin  will  occur.  The  best  results  are  obtained  if  the  tempera- 
ture of  the  room  is  not  less  than  15°  C. ;  in  a  cold  room  no  result 
is  usually  gotten.  To  purify  the  crystals,  a  feebly  acid  solution  of 
ammonium  sulpliate  is  used,  crystallization  being  hastened  by  inocu- 
lation with  a  few  crystals  of  the  substance.  After  standing  until 
crystals  cease  to  separate  out,  and  after  they  have  been  removed, 
the  remaining  solution  is  dialyzed  against  running  Avater  until  the 
sulphuric  acid  has  been  almost  entirely  removed.  It  is  then  heated 
on  a  water-bath  until  all  coagulable  material  has  separated  out. 
This  is  filtered  off  and  thoroughly  washed  with  hot  water  until  the 
filtrate  is  free  from  sulphuric  acid,  and  no  longer  gives  a  cloud  with 
phosphotungstic  acid.  The  resulting  substance  is  dried  at  110°  C. 
and  represents  the  conalbumin. 


THE  REPRODUCTIVE  GLANDS.  459 

To  isolate  the  euglobulin  from  the  total  globulin  fraction,  this  is 
repeatedly  washed  with  a  one-half  saturated  solution  of  ammonium 
sulphate  (by  centrifugation)  until  the  salt  solution  no  longer  gives 
the  biuret  reaction.  The  euglobulin  is  finally  collected  on  a  filter, 
coagulated  in  the  drying-oven  at  100°  C,  and  washed  with  hot 
distilled  water  until  free  from  salts. 

Ovomucoid. — The  mucoid  substance  wdiich  can  be  isolated  from 
the  albumen  of  hens'  eggs  is  present  in  considerable  amount,  consti- 
tuting about  10  per  cent,  of  the  total  solids.  Elementary  analysis 
of  ovomucoid  has  given  the  following  results  :  C  =  48.82,  H  = 
6.96,  N  =12.51,  S  =  2.19.  The  greater  part  of  the  sulphur  (not 
less  than  three-fourths)  is  present  in  loosely  combined  form.  On 
boiling  with  dilute  mineral  acids  it  yields  a  reducing  substance — 
glucosamin.  A  chondroitin-sulphuric  acid  complex  is  not  present 
in  the  ovonuicoid  molecule. 

According  to  most  authors,  the  ovomucoid  does  not  give  the 
Adarakiewicz  reaction,  while  Langstein  states  that  in  the  case  of 
his  own  preparations  he  always  obtained  a  positive  result.  He 
suggests  that  the  negative  findings  of  others  may  have  been  refera- 
ble to  the  possible  absence  of  glyoxylic  acid  in  the  glacial  acetic 
acid  employed. 

The  substance  cannot  be  precipitated  with  the  common  mineral 
acids,  acetic  acid,  and  potassium  ferrocyanide,  nor  by  salting  with 
sodium  chloride,  magnesium  sulphate,  or  sodium  sulphate.  Tan- 
nic acid,  phosphotungstic  acid,  ammoniacal  subacetate  of  lead 
solution,  alcohol,  and  ammonium  sulphate,  when  added  to  satura- 
tion, cause  the  substance  to  separate  out.  It  is  soluble  in  water, 
and  is  not  coagulated  by  boiling.  On  evaporating  its  solutions  to  dry- 
ness it  is  rendered  insoluble  in  cold  water,  but  dissolves  on  boiling. 

IsoLATiox. — To  isolate  the  ovomucoid,  the  albumen  is  diluted 
with  water,  as  above,  slightly  acidified  with  acetic  acid,  and  boiled. 
The  coagulable  albumins  are  thus  coagulated  and  filtered  off.  The 
filtrate,  which  still  gives  the  biuret  reaction,  owing  to  the  presence 
of  the  mucoid,  is  concentrated  and  precipitated  with  alcohol,  or 
saturated  with  ammonium  sulphate.  The  mucoid  is  filtered  off  and 
can  then  be  purified  by  repeated  solution  in  water  and  reprecipita- 
tion  with  alcohol. 

The  Yolk. — The  yolk  of  the  egg  represents  the  ovum  proper.  It 
is  surrounded  by  a  delicate  membrane — the  membrana  pellucida — 
which  supposedly  consists  of  keratin  or  a  closely  related  substance. 
Owing  to  the  extensive  development  of  the  protoplasmic  portion  of 
the  cell  proper,  the  germinal  vesicle  is  found  at  the  extreme  periph- 
ery of  the  yolk,  immediately  beneath  the  limiting  membrane.  It 
occupies  the  centre  of  the  discus  proligerus  or  cicatricula,  which 
rests  upon  a  flask-like  cavity  with  a  long,  narrow  neck  that  extends 
to  the  centre  of  the  yolk,  and  is  occupied  by  the  so-called  vJiite  yolk. 
This  surrounds  the  cicatricula  and  also  forms  a  layer  along  the  pe- 
riphery of  the  yolk,  immediately  beneath   the  vitelline  membrane. 


460  THE  GLANDULAR   ORGANS. 

It  contains  albumins,  nuoleins,  lecithins,  potassium  salts,  and  possibly 
also  traces  of  glycogen,  though  this  is  douljtfnl. 

AVhen  broken,  the  yolk  constitutes  a  creamy,  viscid  material,  of 
an  orange-yellow  color,  M'hich  forms  an  emulsion  with  water,  and  is 
coagulated  by  alcohol  and  on  boiling.  Its  reaction  is  feebly  alka- 
line. On  microscopical  examination  it  is  seen  to  consist  of  innumer- 
able spherules,  some  of  which  are  rich  in  fats  and  lipochromes, 
while  others,  which  are  smaller,  are  colorless,  transparent,  semi- 
crystalline  structures  of  an  albuminous  character.  In  the  eggs  of 
certain  amphibia  and  fishes  distinctly  crystalline  bodies  are  further 
met  with,  which  are  sjioken  of  as  yolk  platelets,  and  are  analogous 
to  the  aleuron  granules  of  seeds.  As  has  already  been  mentioned, 
they  probaV)ly  consist  of  a  compound  of  albumins  with  lecithins  and 
nucleins.  The  ichthklin,  which  is  found  in  carp  eggs,  and  which  in 
amorphous  form  is  known  as  ichthulin,  belongs  to  this  category.^ 
The  same  holds  good  of  the  ichthin  of  shark  eggs  and  the  emydin 
of  tortoise  eggs. 

As  I  have  stated,  the  yolk  of  hens'  eggs  represents  about  29  per 
cent,  of  the  entire  weight.  Its  actual  weight  may  thus  vary  between 
8.7  and  20.3  grammes.  The  general  composition  of  the  yolk  is  seen 
in  the  following  analyses,  which  are  taken  from  Gautier  : 

Per  cent. 

"Water 47.19-51.49 

Solids 48.51-42.81 

Fats  (olein,  palmitlu,  aud  stearin) 21.30-22.84 

Vitellin  and  other  albumins 15.G3-15.76 

Lecithins 8.43-10.72 

Cholesterin 0.44-  1.75 

C'erebrin 0.30 

Mineral  salts 3.33-  0.36 

Coloring-matter  \  ^  -.^ 

Glucose  i      

Analysis  of  the  mineral  salts,  calculated  for  100  parts  of  ash,  has 
given  the  following  results  (Poleck  and  AVeber) : 

Sodium  (XajO) 5.12-  6.57 

Potassium  (K,0) 8.05-  8.93 

Calcium  (CaO) 12.21-13.28 

Magnesium  (MgO)      2.07-  2.11 

Iron  (FeA) 1.19-  1.45 

Phosjjhoric  acid,  free  (P2O5) 5.72 

Phosphoric  acid,  combined 63.81-66.70 

Silicic  acid  (SiO^) 0.55-  1.40 

Chlorine traces 

1  From  recent  researches  of  Levene  it  appears  that  different  forms  of  iclithuliii 
exist.  The  ichthulin  of  carp  eggs  thus  yields  a  reducing  substance  on  hydrolytic 
decomposition,  while  that  of  the  cod  apparently  contains  no  carbohydrate  radicle. 
The  latter,  on  treating  with  alkalies,  yields  a  ))aranucleinic  acid,  which  is  similar 
to  vitellinic  acid  (see  below).  Elementary  analysis  of  the  two  forms  has  given  the 
following  results:  Ichthulin  of  carp  egg^  (Walter):  C,  53.52  ;  H,  7.6;  X,  15.63; 
S,  0.41 ;  P,  0.43  ;  Fe,  0.10  ;  O,  22.19  per  cent.  Ichthulin  of  codfish  eggs  :  C,  52.44  : 
H,  7.45  ;  N,  15.96  ;  S,  0.92  ;  P,  0.65  ;  Fe  and  O,  22.58  per  cent. 


THE  REPRODUCTIVE  GLANDS.  461 

Of  the  mineral  constituents,  the  large  amount  of  calcium  and 
phosphoric  acid  is  especially  noteworthy.  Soluble  phosphates, 
however,  are  not  found  as  such  in  the  yolk.  The  amount  of  potas- 
sium and  sodium,  it  will  be  observed,  is  much  smaller  than  in  the 
albumen. 

The  Albumins. — Our  knowledge  of  the  individual  albumins  which 
are  found  in  the  yolk  is  still  very  imperfect.  But  it  appears  from 
recent  researches  that  they  are  represented  notably  by  nucleo-albu- 
mins,  which  in  turn  may  be  combined  with  lecithins  to  form  com- 
plex lecithalbumins.  This,  however,  is  not  proved,  and  it  is  as- 
sumed by  some  that  the  lecithins  which  are  obtained  so  commonlv 
together  with  the  albumins  do  not  exist  in  chemical  combination., 
but  are  to  be  regarded  as  contaminations.  The  best  known  repre- 
sentative of  the  nucleo-albumins  of  the  yolk  is  the  so-called  ovo- 
vitellin.    True  nucleins  do  not  occur  in  the  yolk. 

Ovovitellin. — Formerly  this  was  regarded  as  a  globulin,  but  it  is 
now  known  to  be  a  nucleo-albumin  in  which  an  albuminous  radicle 
is  combined  with  a  paranuclein — i.  e.,  a  nuclein  which  does  not  yield 
nucleinic  bases  on  decomposition  with  mineral  acids.  The  substance 
has  thus  far  not  been  obtained  free  from  lecithins,  and  it  is  for  this 
reason  that  the  latter  is  thought  by  some  to  be  present  in  chemical 
combination. 

Of  the  character  of  the  albuminous  radicle  which  is  present 
in  combination  with  the  paranuclein  nothing  is  known.  The 
paranuclein  has  recently  been  studied  in  detail  by  Levene  and 
Alsberg.  They  term  it  avivitellinic  acid,  and  give  the  following 
figures  to  express  its  elementary  composition  :  C,  32.31  ;  H,  5.58 ; 
^,  13.13  ;  P,  9.88 ;  S,  0.3266  ;  O,  38.28  per  cent.  In  addition 
they  found  0.57  per  cent,  of  iron,  which  is  present  in  organic  com- 
bination. This  is  especially  interesting  in  view  of  the  fact  that 
Bunge  also  obtained  a  nuclein  from  the  yolk  of  hens'  eggs,  which 
contained  iron,  and  which  he  termed  hcematogen,  as  the  product  must 
of  necessity  be  concerned  in  the  formation  of  the  blood-coloring  mat- 
ter of  the  developing  animal.  The  elementary  composition  of  Bunge's 
haematogen,  however,  is  different  from  Levene's  avivitellinic  acid, 
viz.,  C,  42.11;  H,  6.08;  N,  14.73;  S,  0.55;  P,  5.19;  Fe,  0.29 ; 
and  O,  31.05  per  cent.  Its  relation  to  ovovitellin  is  at  present 
not  clear,  but  it  is  manifestly  closely  related  to  avivitellinic  acid. 

The  albuminous  radicle  of  avivitellinic  acid  manifestly  contains 
the  protamin  group,  as  Levene  was  able  to  isolate  both  arginin  and 
histidin  from  its  decomposition-products,  which  resulted  on  boiling 
the  substance  for  seventy-two  hours  with  a  20  per  cent,  solution  of 
hydrochloric  acid.  Whether  or  not  lysin  is  also  present  remains  to 
be  seen.  The  amount  of  arginin  and  histidin  obtained  was  so  small, 
however,  that  it  is  scarcely  warrantable  to  assume  that  a  protamin 
constitutes  the  entire  albuminous  radicle,  as  in  the  case  of  the  nu- 
cleins which  can  be  obtained  from  certain  fishes.  The  substance 
gives  Millon's  reaction,  moreover,  which  is  not  obtained  with  pro- 


462  THE  GLANDULAR  ORGANS. 

tamins.  The  biuret  reaction  was  positive.  For  a  more  detailed 
account  of  Levene's  most  interesting  work,  and  a  description  of  the 
method  which  was  employed  for  isolating  the  avivitellinic  acid,  I 
must  refer  the  reader  to  his  article.' 

The  ovivitellin  as  it  is  obtained  from  the  yolk  contains  about  25 
per  cent,  of  lecithin.  It  is  soluble  in  dilute  solutions  of  the  neutral 
salts,  and  in  very  dilute  (1  pro  mille)  solutions  of  hydrochloric  acid, 
and  the  hydrates  and  the  carbonates  of  the  alkalies.  In  water  it 
is  insoluble,  and  accordingly  is  precipitated  from  its  solutions  on 
copious  dilution.  On  prolonged  contact  with  water  its  properties 
are  changed,  and  it  is  converted  gradually  into  an  albuminate-like 
substance.  Sodium  chloride  when  added  to  saturation  causes  only 
a  partial  precipitation.  When  slowly  heated  in  its  solutions  of 
neutral  salts  it  coagulates  between  70°  and  75°  C. ;  when  rapidly 
heated,  coagulation  is  retarded  until  80°  C.  is  reached.  On  diges- 
tion with  gastric  juice  ovivitellin  yields  a  paranuclein — avivitellinic 
acid.  From  the  ovivitellin  of  the  eggs  of  the  bony  fishes  a  gluco- 
paranuclein  may  be  obtained. 

Elementarv  analvsis  of  the  ichthulin  of  carp  ega:s  has  given  the 
following  results  :  C,  53.52  ;  H,  7.6  ;  X,  15.63  ;  0^22.19  ;  S,  0.41  ; 
P,  0.43  ;  and  Fe,  0.1  per  cent.  For  the  ichthulin  of  codfish  eggs 
Levene  found  C,  52.44 ;  H,  7.45  ;  N,  15.96  ;  S,  0.92 ;  P,  0.65  ;  Fe 
and  O,  22.58  per  cent.  On  treating  with  alkalies  a  substance  is 
obtained  from  this  latter  form,  which  is  quite  similar  in  compo- 
sition to  avivitellinic  acid,  as  is  seen  from  the  figures :  0,  32.56 ; 
H,  6 ;  N,  14.03 ;  S,  0.146 ;  P,  10.34  per  cent.  It  is  termed  ich- 
thulmic  acid  (see  also  page  461). 

Isolation. — To  isolate  the  ovivitellin  from  the  yolk,  it  is  well  to 
employ  a  large  number  of  eggs.  The  yolks  are  thoroughly  mixed 
with  an  equal  volume  of  a  10  per  cent,  solution  of  sodium  chloride, 
and  are  completely  extracted  with  ether,  by  shaking,  viz.,  until  no 
more  coloring-matter  can  be  removed,  and  the  sodium  chloride 
solution  has  become  perfectly  transparent.  This  is  then  diluted 
with  twenty  times  its  volume  of  water,  and  the  ovivitellin  thus 
precipitated.  To  purify  the  substance  further,  it  is  dissolved  re- 
peatedly in  a  10  per  cent,  saline  solution,  and  reprecipitated  with 
water.  It  is  washed  finally  with  alcohol  and  ether,  and  dried  over 
sulphuric  acid. 

The  Fats. — The  fat  of  the  yolk  consists  almost  entirely  of  olein, 
])almitin,  and  stearin.  As  a  whole,  it  contains  a  somewhat  smaller 
amount  of  carbon  than  ordinary  fat,  which  may  be  due  to  the  pres- 
ence of  mono-  and  diglycerides,  or  to  the  presence  of  a  fatty  acid 
which  contains  less  carbon  than  usual.  On  saponification  Liebcr- 
mann  obtained  40  per  cent,  of  oleic  acid,  38.04  per  cent,  of  palmitic 
acid,  and  15.21  per  cent,  of  stearic  acid. 

The  lipochromes  or  luteins  of  the  yolk  can  be  isolated  as  follows : 
the  fats  of    the  yolk  are  saponified  by  boiling  with  an  alcoholic 
^  Zeit.  f.  physiol.  Chem.,  vol.  xxxi.  pp.  543-556. 


THE  REPRODUCTIVE  GLANDS.  463 

solution  of  sodium  hydrate.  The  alcohol  is  then  evaporated.  The 
remaining  solution  is  treated  with  calcium  chloride,  which  trans- 
forms the  soluble  sodium  salts  into  the  corresponding  insoluble  cal- 
cium salts.  On  cooling,  the  soaps  are  extracted  with  petroleum- 
ether,  .which  takes  up  the  lipochromes.  On  evaporation  they  are 
then  obtained  in  pure  form.  The  entire  process  of  isolation  must 
be  carried  on  in  the  absence  of  daylight,  as  otherwise  the  pigments 
are  decomposed  after  being  separated  from  the  fats.  In  birds'  eggs 
a  yellow  lipochrome,  vitelloiutciu,  is  notably  found,  but,  in  addi- 
tion, traces  of  a  red  pigment  of  the  same  order,  which  is  termed 
vitcUorubin ,  may  also  be  encoinitered.  This  latter  cannot  well  be 
obtained  by  extracting  the  soaps  with  petroleum-ether  directly,  but 
it  is  necessary  pi'eviously  to  decompose  these  with  a  mineral  acid. 

Lecithins. — The  general  properties  of  the  lecithins  have  been  con- 
sidered in  a  previous  chapter  (page  78). 

IsOLATiox. — To  isolate  the  lecithins,  the  method  of  Ziilzer  may 
be  conveniently  employed.  To  this  end,  the  yolks  of  a  large  num- 
ber of  eggs  (fifty  or  more)  are  first  extracted  with  ether  by  shaking, 
until  the  ethereal  solution  takes  up  no  more  pigment.  The  ethereal 
extracts  are  united,  the  ether  is  distilled  off,  and  the  oil  filtered  off 
at  the  temperature  of  the  body.  This  is  best  accomjjlished  in  a 
thermostat.  The  yellow,  somewhat  frothy  material  which  remains 
on  the  filter  is  dissolved  in  as  little  ether  as  possible,  and  precipi- 
tated with  acetone.  The  precipitate  is  collected  on  a  filter  and 
washed  with  acetone  until  the  wash-acetone  dissolves  no  more 
cholesterin.  The  residue  is  again  dissolved  in  a  small  amount  of 
ether  or  benzol.  To  this  solution  an  excess  of  absolute  alcohol  is 
added,  when  on  standing  a  white  amorphous  substance  separates 
out,  which  can  be  obtained  in  crystalline  form  by  solution  in  hot 
alcohol  and  cooling ;  this  apparently  consists  of  tripalmitin.  After 
filtration  the  pure  lecithin  can  then  be  obtained  from  the  ether- 
alcoholic  solution  by  precipitating  with  acetone,  as  before,  or  by  dis- 
tillino-  oif  the  alcohol  and  ether.  The  resulting:  material  is  dried  in 
the  vacuum.  Its  phosphorus  varies  between  3.7  and  4.1  per  cent, 
in  amount. 

Incubation. — Of  the  chemical  changes  which  take  place  during 
the  process  of  fertilization,  and  in  which  the  nucleus  of  the  ovum  is 
primarily  concerned,  we  know  nothing.  But  there  can  be  no  doubt 
that,  in  fishes  at  least,  the  protamin  radicle  of  the  nucleins  of  the 
spermatozoa  plays  an  important  part.  As  a  result,  the  reproductive 
function  of  the  ovum,  which  previously  has  remained  dormant, 
now  manifests  itself  in  the  mysterious  morphological  changes  which 
the  cell  undergoes,  and  which  end  in  the  production  of  an  organism 
that  is  morphologically  and  chemically  like  its  2)arents. 

In  mammals  the  food-stuffs  which  are  required  by  the  devel- 
oping organism  are  constantly  sujiplied  throiigh  the  blood  of  the 
mother-animal,  but  in   the  lower  forms  of  life  they  are  furnished 


464  THE  GLANDULAR   ORGANS. 

directly  in  the  egg  itself.  These  products  have  been  studied  in 
some  detail  in  the  foregoing  pages,  and  we  have  seen  that  they  are 
in  part,  at  least,  specific  of  the  egg,  and  do  not  occur  elsewhere  in 
the  animal  body.  This  holds  good  more  especially  of  the  albu- 
mins, and  it  follows  that  all  those  forms  that  enter  into  the  com- 
position of  the  various  tissues  must  of  necessity  be  produced  from 
the  pre-existing  forms  during  the  development  of  the  young  animal. 
The  fats  may,  in  part,  be  utilized  directly  in  the  construction  of  the 
fats  of  the  embryo,  but  to  a  large  extent,  no  doubt,  they  represent 
the  principal  form  of  energy  which  is  placed  at  the  disposal  of  the 
developing  organism.  Carbohydrates,  as  such,  are  practically  lack- 
ing among  the  food-stuifs  of  the  egg,  and  must  hence  be  formed 
synthetically.  That  glycogen  can  be  demonstrated  in  the  tissues 
of  the  embryo  at  a  very  early  date,  has  already  been  stated,  which 
proves  in  itself  that  the  animal  organism  is  not  dependent  upon 
the  ingested  carbohydrates  for  its  glycogen  supply.  As  nuclear 
nucleins,  moreover,  do  not  occur  in  the  egg,  it  follows  that  these 
also  must  be  formed  from  other  albuminous  substances,  and  there 
can  be  little  doubt  that  the  paranucleins  are  here  of  prime  impor- 
tance. In  this  connection  it  is  interesting  to  note,  however,  thpt 
Mesernitzki  found  xanthin-bases  in  identical  amounts  in  non-incu- 
bated eggs  as  in  early  chick-embryos.  The  salts  which  are  required 
by  the  developing  organism  are,  as  has  been  seen,  present  in  the 
egg  in  abundance. 

The  essential  factor,  however,  which  is  necessary  to  development 
after  fertilization,  is  an  abundant  supply  of  oxygen,  and  a  tempera- 
ture of  about  40°  C.  The  requisite  amount  of  oxygen  is  obtained 
from  the  air  by  a  process  of  ditfusion  through  the  shell.  In  return 
carbon  dioxide  is  eliminated,  together  Avith  a  small  amount  of 
nitrogen.  These  respiratory  changes  are  but  slight  in  the  begin- 
ning, but  gradually  increase.  Water  also  is  given  off,  and,  as  a 
result,  the  weight  of  the  egg  diminishes.  The  increase  of  the  solids 
in  the  developing  animal  is,  of  course,  accompanied  by  a  correspond- 
ing diminution  of  those  of  the  egg  itself. 

Systematic  chemical  examinations  of  the  ovum  in  its  various 
stages  of  development  have  thus  far  not  been  made.  Liebermann 
appears  to  be  the  only  one,  indeed,  who  has  attempted  the  problem. 
His  principal  results  may  be  summarized  as  follows  :  during  the 
first  stage  of  development  tissues  are  formed,  which  are  very  rich  in 
water ;  later,  however,  the  amount  of  water  decreases.  The  abso- 
lute amount  of  substances  which  are  soluble  in  water  steadily  in- 
creases, while  their  relative  amount  diminishes  as  compared  with 
the  remaining  solids.  After  the  fourteenth  day  a  large  increase  in 
the  amount  of  fat  is  noted,  while  previously  this  remains  fairly  con- 
stant. The  amount  of  soluble  albumins  and  albuminoids  increases 
steadily  and  in  such  a  manner  that  the  absolute  quantity  increases, 
while  their  relative  amount  remains  nearly  constant.  Up  to  the 
tenth  day  no  collagen  is  found,  but  after  the  fourteenth  day  a  sub- 


THE  REPRODUCTIVE  GLANDS.  465 

stance  is  present  which  on  boiling  with  water  yields  a  material  sim- 
ilar to  cartilaginous  gliitin.  A  mucinous  substance  is  found  about 
the  sixth  day,  but  it  subsequently  disappears.  The  amount  of 
hseraoglobin  steadily  increases  in  its  relation  to  the  body-weight. 

Levene  has  recently  shown  that  incubated  eggs  contain  raono- 
amido-acids,  and  he  regards  it  as  probable  that  the  preparations 
which  he  obtained  consisted  of  an  equimolecular  mixture  of  mono- 
araido-butyric  acid  and  mono-amido-valerianic  acid.  The  results 
were  obtained  with  eggs  that  were  twenty-four  hours  and  seven 
days  old. 

Interesting  also  are  Levene's  observations  on  the  amount  of  albu- 
mins at  different  stages  of  development,  which  were  made  on  fish 
eggs.  Non-incubated  eggs  contained  66  per  cent,  of  albuminous 
nitrogen  ;  after  twenty-four  hours'  incubation,  53.57  per  cent,  was 
found  ;  after  ten  days,  64.79  per  cent ;  and  at  the  expiration  of 
nineteen  days,  71.84  per  cent.  During  this  period  there  was  also  a 
steady  increase  in  the  amount  of  nitrogen  referable  to  non-albu- 
minous substances  that  could  be  precipitated  with  phosphotungstic 
acid,  viz.,  from  12.07  to  28.25  per  cent. 

On  the  basis  of  Spitzer's  observations  on  the  nature  of  the  oxida- 
tion-ferments as  nucleoproteids  Liib  expressed  the  opinion  that  the 
nucleus  represents  the  organ  of  oxidation  of  living  matter,  and  that 
cellular  matter  devoid  of  nuclei  is  incapable  of  regenerating  its  kind 
for  the  reason  that  its  power  of  oxidation  is  too  insignificant. 
Jacoby,  however,  has  shown  that  in  pig  embryos,  at  least  at  a  time 
when  the  first  indications  of  the  formation  of  a  bony  skeleton  exist, 
aldehydase  at  any  rate  cannot  be  demonstrated.  Whether  or  not 
it  is  present  as  a  zymogen,  has  not  been  investigated.  Later  the 
aldehydase  appears,  viz.,  in  embryos  which  are  9  cm.  long  and 
longer.  A  tangible  basis  for  Lob's  assumption  hence  does  not 
exist. 

The  chemical  composition  of  the  allantoic  fluid  and  the  amniotic 
fluid  has  already  been  considered  (page  370). 

The  placenta  has  not  as  yet  been  studied  in  detail,  but  it  is  likely 
that  its  greater  portion  consists  of  collagen,  in  accordance  with  its 
fibrous  structure.  In  its  marginal  zone  two  pigments  have  been 
encountered  which  apparently  are  related  closely  to  bilirubin  and 
biliverdin,  and  are  derivatives  of  hemoglobin.  The  orange  pigment 
may  be  obtained  in  crystalline  form,  while  the  green  pigment,  which 
has  been  termed  hoematochlovine,  is  amorphous. 

30 


CHAPTER   XXII. 

THE  DUCTLESS  GLANDS. 

THE  THYROID  GLAND. 

Of  the  function  of  the  thyroid  gland  little  is  known.  Removal 
of  the  organ  gives  rise  to  serious  symptoms  which  may  be  of  a 
chronic  or  an  acute  character.  In  the  first  instance  there  is  evi- 
dence of  marked  impairment  of  the  general  metabolic  functions  and 
of  heat-production,  associated  with  impairment  of  muscular  and 
mental  power  (mvxoedema),  and  if  occurring  in  children  there  is 
marked  retardation  in  grow'th  (cretinism).  When  of  an  acute  char- 
acter tetanic  symptoms  commonly  develop.  When  removal  is 
complete,  death  sooner  or  later  results.  If,  however,  the  destruc- 
tion or  removal  of  the  gland  is  partial,  deleterious  results  do  not 
necessarily  follow.  It  is  thus  manifest  that  the  function  of  the 
organ  is  a  most  important  one,  and  it  is  interesting  to  note  that  the 
apparent  antitoxic  properties  of  the  gland  persist  even  after  its 
removal  from  the  body  and  subsequent  desiccation.  The  various 
symptoms  which  have  just  been  described  as  following  extirpation 
of  the  organ  may  thus  be  prevented  by  administration  of  the 
dried  gland,  and  in  cases  of  atrophy  a  curative  effect  may  similarly 
be  obtained.  It  is  noteworthy,  moreover,  that  the  administration 
of  the  substance  has  a  marked  effect  upon  the  nitrogenous  metabo- 
lism of  the  body,  which  is  distinctly  increased,  and,  if  continued, 
emaciation  results  although  an  abundant  amount  of  food  is  ingested, 
and  digestion  and  resorption  remain  unimpaired.  In  some  instances 
true  diabetes  develops.  In  addition,  an  increased  pulse-rate  and 
heightened  blood-pressure  are  commonly  observed,  and  even  sud- 
den death  may  occur  during  the  use  of  the  substance. 

That  these  curious  properties  of  the  thyroid  gland  shonld  also 
find  expression  in  its  chemical  comjiosition  suggests  itself  at  once. 
Numerous  attempts  have  accordingly  been  made  to  isolate  the 
''active  principle"  of  the  organ,  and  to  a  certain  extent  these  at- 
tempts have  been  successful.  Baumann  and  Roos  thus  succeeded 
in  isolating  from  the  gland  a  substance  which  has  manifestly  the 
same  properties  as  the  entire  organ  in  preventing  the  deleterious 
results  which  follow  its  extirpation  or  atrophy,  and  which  also  has 
the  same  effect  upon  the  circulation  and  the  nitrogenous  metabolism. 
This  substance  Baumann  termed  thijroiodine,  from  the  fact  that  it 
contains  iodine  in  organic  combination.    It  is  obtained  by  boiling  the 

4fi6 


THE  THYROID   GLAND.  467 

gland  for  several  hours  with  a  10  per  cent,  solution  of  sulphuric 
acid,  and  by  subsequently  extracting  the  insoluble  residue  with  90 
per  cent,  alcohol.  It  is  insolul)le  in  water  and  acids,  but  readily 
dissolves  in  dilute  alkaline  solutions,  from  which  it  is  precipitated 
by  adding  an  excess  of  an  acid.  The  substance,  as  first  obtained  by 
Bauniann,  contained  9.3  per  cent,  of  iodine  and  a  small  amount  of 
phosphorus.  This  latter,  however,  he  regarded  as  a  contamination, 
and  he  expressed  the  opinion  that  future  researches  would  show  that 
the  chemically  pure  body  contained  even  more  iodine  than  the  crude 
product  he  obtained.  Regarding  the  chemical  nature  of  the  thy- 
roiodine,  which  itself  does  not  give  the  biuret  reaction,  Baumann 
supposed  that  it  existed  in  the  gland  in  combination  with  an  albu- 
min, viz.,  as  a  thyro-iodoglobulin-  or  albumin.  This  has  since 
been  proved,  through  the  researches  of  Oswald,  who  succeeded  in 
extracting  from  the  gland  a  globulin  which  contains  the  entire 
amount  of  iodine,  and  has  all  the  physiological  properties  of  the 
thyroiodine  (iodotliyrin).  It  yields  Baumann's  thyroiodine  on 
decomposition  with  mineral  acids.  This  substance  is  termed  thy- 
reoglobulin. Together  witii  another  albuminous  substance  belonging 
to  the  nucleoproteids  the  thyreoglobulin  forms  the  colloid  substance 
of  tlie  gland. 

Thyreoglobulin. — The  quantity  of  thyreoglobulin  is  directly 
dependent  upon  the  amount  of  the  colloid,  and  is  thus  subject  to 
variation.  Normal  glands  contain  on  an  average  from  1  to  8 
grammes  of  the  dried  substance.  Much  larger  amounts,  viz.,  50  to 
100  grammes,  have  been  observed  in  goitres  that  were  especially 
rich  in  colloid.  In  sheep  it  has  been  found  in  the  proportion  of 
1  :3,  or  2  :3,  as  compared  witii  the  total  amount  of  solids,  and  simi- 
lar results  have  been  obtained  in  the  pig.  Its  general  elementary 
composition  in  animals  of  the  same  species  is  quite  constant,  and 
varies  but  little  indeed  in  animals  of  different  species.  The 
amount  of  iodine,  however,  which  is  present  in  organic  combination 
is  subject  to  fairly  wide  variations.  This  is  shown  in  the  follow- 
ing analyses,  which  are  taken  from  Oswald  : 

Pig.  Sheep.  ox.  ^'™l-     Colloid^goitre. 

C      52.21  52.32  52.45  51.85  52.02 

H 6.83  7.02  6.93  6.S8  6.91 

N 16.59  15.90  15.92  15.49  15.32 

I      0.46  0.39  0.86  0.2-0.3  0.04-0.09 

S 1.86  1.95  1.83  1.87  1.93 

O 22.15  22.42  22.01  23.57  23.75 

It  is  thus  seen  that  in  colloid  goitres  especially  small  amounts  of 
iodine  are  apparently  ])resent.  And  it  appears  that  the  amount  of 
iodine  is  the  lower,  the  more  advanced  the  colloid  degeneration — 
i.  e.,  the  larger  the  amount  of  thyreoglobulin.  Oswald  explains 
this  observation  by  assuming  the  simultaneous  existence  in  goitres 
of  an  iodized  and  a  non-iodized  globulin.  The  iodized  form  is  found 
only  in  glands  which  contain   colloid,  while  it  is  never  found  in 


468  THE  DUCTLESS  GLANDS. 

thyroids  that  are  free  from  colloid,  such  as  jxarenchymatons  goitres 
and  the  j^lands  of  tlic;  newborn.  Oswald  concludes  that  the  thyreo- 
globulin is  only  iodized  after  it  leaves  the  follicle-cells.  At  the 
same  time  it  is  noteworthy  that  non-iodized  thyreoglobulin  alone 
never  leaves  the  cells,  but  only  if  iodothyreoglobulin  is  simultane- 
ously secreted.  The  amount  of  iodine  m  a  gland  can  be  artificially 
increased  by  the  ingestion  of  iodine  or  iodides  as  such,  and  it  is  to 
he  noted  that  the  physiological  activity  of  the  gland  can  thus  be  in- 
creased, while  this  is  not  possible  by  iodizing  the  thyreoglobulin  in 
vitro. 

The  existence  of  a  non-iodized  thyreoglobulin  is  especially 
interesting  in  view  of  the  fact  that,  whereas  the  iodized  globulin 
possesses  all  the  specific  properties  of  the  entire  gland,  the  non- 
iodized  substance  is  inert.  An  adequate  explanation  of  this  curious 
phenomenon  cannot  at  present  be  given,  but  we  may  imagine  that 
in  those  instances  in  which  the  iodine  is  normally  absent  its  place 
may  be  taken  by  some  other  halogen,  or  a  compound  halogen  which 
has  escaped  observation.  To  conclude  that  the  iodized  substance 
is  not  the  active  principle  of  the  gland,  on  the  basis  that  the 
glands  of  some  animals  contain  no  iodine,  and  that  its  amount  is 
more  or  less  variable  and  can  artificially  be  increased,  is  scarcely 
warrantable.  It  is  conceivable  that  in  sucklings,  for  example,  in 
which  iodine  is  commonly  absent,  a  compound  halogen  takes  its 
place,  and  is,  for  the  time  being  at  least,  fully  capable  of  preventing 
the  development  of  the  complex  of  symptoms  which  Me  term  ca- 
chexia strumijwiva . 

In  its  general  ]>roperties  thyreoglobidin  resembles  the  common 
globulin  of  the  blood.  It  differs,  from  this,  however,  in  the  fact 
that  it  is  precipitated  from  its  saline  solutions  on  the  addition  of 
dilute  acids.  In  this  respect  it  resembles  the  myosin  of  muscle- 
tissue.  Its  point  of  coagulation,  in  a  10  per  cent,  solution  of  mag- 
nesium sulphate,  varies  between  65°  and  67°  C  It  is  precipitated 
from  its  solutions  by  the  addition  of  an  equal  volume  of  a  saturated 
solution  of  ammonium  sulphate. 

On  decomposition  with  dilute  acids  it  yields  the  thyroiodine  of 
Baumann,  but  contains  more  iodine,  viz.,  14.29  per  cent.,  and,  like 
its  mother-substance,  it  is  free  from  phosphorus.  On  further 
decomposition  it  loses  its  activity  altogether. 

Thyreo-nucleoproteid. — A  nucleoproteid  is  found  in  associa- 
tion with  thyreoglobulin  in  the  colloid  material  of  the  gland,  but 
is  present  in  much  smaller  amounts  ;  it  contains  0.16  per  cent,  of 
phosphorus.  In  a  10  per  cent,  solution  of  magnesium  sulphate  it 
coagulates  at  73°  C.  On  digestion  with  gastric  juice  a  nuclein  is 
split  off,  which  contains  xanthin-bases.  The  substance  is  free  from 
iodine,  and  is  physiologically  inert.  It  is  ]>recipitated  from  its 
solutions  by  salting  with  ammonium  suljihate  to  saturation. 

For   a    further    description    of  the    two    albumins,    and   of  the 


THE  ADRENAL   GLANDS.  469 

methods  which  may  be  employed  for  tlieir  isolation,  the  reader  is 
referred  to  Oswald's  paper.^ 

The  extractives  of  the  thyroid  gland  are  represented  by  traces 
of  xanthin,  hypoxanthin,  leucin,  succinic  acid,  and  paralactic  acid. 
In  addition,  notable  quantities  of  kreatinin  may  be  obtained. 

Among  the  mineral  constituents  of  the  thyroid  gland  the  presence 
of  traces  of  arsenic  is  of  interest ;  according  to  Gautier  and  Bert- 
rand,  it  represents  a  normal  and  constant  component  of  the  gland. 

THE  ADRENAL  GLANDS. 

Of  the  function  of  the  adrenal  glands,  nothing  definite  is  known- 
Their  integrity,  however,  is  essential  to  life,  and,  as  in  the  case  of  the 
thyroid,  their  removal  leads  to  the  death  of  the  animal.  It  has  been 
noted,  moreover,  that  the  injection  of  blood  from  a  dog  which  has 
died  as  a  result  of  the  operation,  into  the  circulation  of  a  second 
animal  that  has  been  operated  in  the  same  manner,  will  hasten  the 
fatal  end,  while  in  normal  dogs  no  deleterious  results  are  observed. 
It  has  hence  been  concluded  that  the  glands  normally  furnish  a 
secretion  which  renders  certain  metabolic  products  innocuous,  and 
that  the  fatal  result  which  follows  the  removal  of  the  organs  is  the 
result  of  an  auto-intoxication. 

In  man,  disease  of  the  adrenal  glands  leads  to  the  complex  of 
symptoms  which  is  commonly  known  as  Addison's  disease,  and  like- 
wise results  in  death  ;  but  as  in  the  case  of  the  thyroid  gland,  it  has 
been  observed  that  the  fatal  issue  may  here  also  at  least  be  retarded 
by  the  administration  of  an  aqueous  extract  of  the  organs.  Experi- 
ments with  such  extracts  have  further  shown  that  the  gland  contains 
a  substance  which  has  a  very  marked  eifect  upon  the  blood-pressure, 
raising  this  far  beyond  the  normal.  This  substance  is  found  in  the 
medullary  portion  of  the  glands.  Further  investigations  have  then 
demonstrated  the  existence  of  a  chromogen  which  on  exposure  to  the 
air  in  aqueous  solution  yields  a  beautiful  carmin-colored  pigment, 
which,  like  its  mother-substance,  is  soluble  in  water.  The  same 
result  is  reached  at  once  on  treating  with  chlorine-,  bromine-,  or 
iodine-water.  This  chromogen  is  present  in  the  intracellular  fluid  of 
the  medullary  portion  of  the  gland.  When  this  is  extracted  with  a 
dilute  acid,  a  violet-red  precipitate  results  on  the  addition  of  an 
excess  of  ammonia,  which  suggests  that  the  pigment  is  of  a  basic 
nature. 

With  a  solution  of  ferric  chloride  the  juice  that  can  be  expressed 
from  the  glands  gives  a  bright  emerald-green  color.  This  reaction 
has  been  referred  to  the  supposed  presence  of  pyrocatechin,  but  thus 
far  this  has  never  been  isolated. 

Modern  researches  lead  to  the  conclusion  that  the  blood-pressure- 
raising  constituent  of  the  gland,  as  also  the  chromogen,  which  gives 
rise  to  the  carmin  color  and  the  pyrocatechin  reaction,  are  identical 
^  Zeit.  f.  pliysiol.  Chem.,  vol.  xxvii.  p.  14. 


470  THE  DUCTLESS  GLANDS 

bodies.  Abel,  M'ho  claims  to  have  isolated  the  blood-pressure-raising 
principle  of  the  glands,  states  that  this  must  in  all  probability  l)e 
classed  with  the  pyrrol  compounds  or  with  the  pyridin  bases  or  alka- 
loids. He  was  unable,  however,  to  obtain  the  free  base,  which  he 
terms  cpinephrin,  in  crystalline  form.  Pyrocatechin  could  not  be 
split  off  from  this  product  on  boiling  with  an  acid,  but  he  states  that 
a  carmin-red  pigment  can  be  separated  from  the  sulphate  of  the 
active  principle  without  destroying  its  power  to  raise  the  blood- 
pressure. 

V.  Fiirth,  who  calls  the  active  principle  suprarenin,  was  likewise 
not  able  to  obtain  it  in  pure  form. 

Later,  Takamine  announced  that  he  had  succeeded  in  isolating 
the  blood-pressure-raising  constituent  of  the  gland  in  a  stable  and 
crystalline  form.     This  substance  he  terms  adrenalin. 

Adrenalin  is  a  light,  white  crystalline  substance,  of  a  slightly 
bitter  taste,  leaving  a  numb  feeling  on  the  tongue  where  it  has  been 
applied.  When  dry,  it  is  perfectly  stable.  On  heating,  it  turns 
brown  at  205°  C.  At  207°  C.  it  melts,  and  is  at  the  same  time 
decomposed.  Its  reaction  is  slightly  alkaline.  In  cold  water  it  is 
soluble  with  difficulty,  but  more  readily  so  in  hot  water.  From  its 
hot  solution  it  crystallizes  out  on  cooling.  It  is  easily  soluble  in 
acids  and  alkalies,  but  not  in  ammonia  or  solutions  of  the  alkaline 
carbonates.  Upon  the  addition  of  ferric  chloride  its  solutions  are 
colored  a  fine  emerald-green,  which  changes  to  a  purple  and  then  to 
a  carmin  red  upon  the  careful  addition  of  caustic  alkali.  Strong  acids 
prevent  the  reaction,  limiting  the  change  of  color  to  a  dirty  yellow- 
ish-green. It  reduces  silver  salts  and  auric  chloride  very  ener- 
getically, while  the  liquid  at  the  same  time  turns  red.  This  also 
occurs  on  treating  with  oxidizing  agents,  such  as  potassium  ferri- 
cyanide  and  potassium  bichromate.  The  usual  alkaloidal  reagents 
do  not  precipitate  the  substance.  With  acids  it  forms  salts,  but 
these  have  not  been  obtained  in  crystalline  form. 

Abel's  most  recent  analysis  of  his  epinephrin  hydrate  only  differs 
from  Takamine's  adrenalin  by  one-half  a  molecule  of  water ;  its 
formula  is  CmHi3N03.|Il20.  Its  structural  composition  is  still 
unknown. 

The  blood-pressure-raising  power  of  adrenalin  is  very  marked. 
The  amount  pro  kilo  of  body- weight  which  is  required  to  raise  the 
blood-pressure  14  Hgmm.  beyond  the  normal  is  one-millionth  part 
of  a  gramme,  and  distinct  physiological  effects  can  be  obtained  by  the 
administration  of  even  one-fourteenth  millionth  part  of  a  gramme. 

Aside  from  the  blood-pressure-raising  properties  the  adrenal 
glands  are  also  of  interest  from  the  fact  that  hypodermic  injec- 
tion of  adrenal  extract  results  in  glucosuria,  even  though  carbohy- 
drates have  been  excluded  from  the  diet  of  the  animal  (dogs).  The 
amount  of  sugar  which  may  ap])ear  is  at  times  considerable,  viz., 
3.8  per  cent.  Analogous  results  are  obtained  during  starvation  and 
at  a  time  when  the  liver  is  free  from  glycogen.     The  glucosuria 


THE  ADRENAL   GLANDS.  471 

persists  for  from  one  to  three  days,  and  is  manifestly  not  dej)endent 
u[)on  clian^es  in  the  hlood-j)hisma.  It  is  noteworthy  that  in  these 
eases  the  elimination  of  niti"o<;en  is  not  increased,  and  that  neither 
acetone  nor  diacetic  aeid  is  found  in  the  urine.  As  ex[)lanation  of 
the  glucosuria  in  these  cases  Blum  is  inclined  to  assume  some 
special  action  on  the  part  of  the  adrenal  extract  upon  the  pancreas 
or  possibly  upon  the  liver.  Similar  results  were  obtained  by  llerter 
and  Wakeman  with  adrenalin  directly.  These  observers  also  found 
that  painting  the  pancreas  with  adrenalin  solution  produced  marked 
glucosuria,  while  this  was  only  slight  if  the  same  was  done  in  the 
case  of  the  liver,  the  spleen,  or  the  brain.  Other  reducing  agents 
j)roduced  a  similar  effect,  so  that  the  authors  concluded  that  a  toxic 
effect  is  exerted  by  all  these  bodies  upon  the  pancreatic  cells  leading 
to  impairment  of  their  oxidi/ing  power,  so  that  normal  coml)Ustion 
of  the  sugar  does  not  occur. 

In  a  number  of  instances  in  which  glucosuria  was  produced  by 
the  injection  of  adrenal  extract,  bile-pigment  also  appeared  in  the 
urine,  but  there  is  no  constant  relation  between  the  two  conditions 
(Blum). 

In  addition  to  these  bodies  the  adrenal  glands  contain  collagen, 
which  enters  into  the  composition  of  the  supporting  tissue  ;  albu- 
mins, which  have  not  been  studied  in  detail ;  and,  further,  a  sub- 
stance which  apparently  is  closely  related  to  jecorin,  and  yields 
fatty  acids,  neurin,  glycerin-phosphoric  acid,  and  glucose  on  hydro- 
lytic  decomposition  with  baryta-water.  Besides,  lecithins  and  a 
small  amount  of  inosit  are  met  Avith.  ])enzoic  acid,  hij^puric 
acid,  and  biliary  acids  are  not  present,  as  was  formerly  sup])osed. 

Jones  has  isolated  a  nucleoj)roteid  from  the  adrenal  gland  which 
seems  to  have  the  same  comiiosition  as  Hanmiarsten's  nucleopro- 
teid  which  he  obtained  from  the  pancreas.  It  is  dextrorotatory 
(«)D  +4:].1°. 

Of  ferments,  the  adrenal  gland  contains  an  aldehydase  (Jacoby) 
which  is  principally  found  in  the  cortex  ;  further,  a  diastase 
(Croftan)  Avhicli  is  capable  of  forming  both  maltose  and  dextrose, 
and  finally  an  autolytic  proteolytic  ferment. 


APPENDIX. 


LABORATORY   EXERCISES. 

Exercise  I. — Prepare  a  1  per  cent.  (0.8  per  cent.)  solution  of 
serum-albumin,  serum-globulin,  and  egg-albumin  in  dilute  saline 
solution.  Determine  the  coagulation-point  of  each,  after  acidifying 
slightly  with  very  dilute  acetic  acid.  To  this  end,  place  the  albu- 
minous solution  in  a  test-tube,  of  which  it  should  fill  about  one- 
third  to  one-half;  clamp  this,  immersed  in  a  beaker  with  water,  and 
fasten  a  thermometer  in  the  albuminous  solution  so  that  the  bulb 
nearly  reaches  the  bottom.  Heat  with  a  small  flame  and  note  the 
temperature  at  which  coagulation  just  begins  ;  continue  to  heat,  and 
finally  boil  the  solution  ;  note  the  changing  appearance  of  the  pre- 
cipitate. Collect  some  of  this  and  attempt  to  dissolve  it  in  water 
or  dilute  saline  solution  ;  then  try  concentrated  acid  and  alkali  with 
the  application  of  heat. 

Study  the  behavior  of  an  albuminous  solution  on  heating  in  the 
presence  of  an  excess  of  dilute  acid  and  alkali  and  in  the  presence 
and  absence  of  salt  (page  30). 

Exercise  II. — 1.  Prepare  a  dilute  solution  of  serum-albumin; 
render  it  feebly  acid  with  acetic  acid ;  coagulate  it  by  boiling ; 
render  the  solution  strongly  acid  with  hydrochloric  acid,  heat,  and 
note  that  the  coagulated  albumin  dissolves;  on  cooling  add  an 
alkali  until  the  reaction  is  only  faintly  acid,  and  heat  again  :  coagu- 
lation does  not  occur. 

2.  Render  a  solution  of  serum-albumin  strongly  alkaline  with 
sodium  hydrate  and  boil:  coagulation  does  not  occur;  then  add 
acetic  acid  to  a  feebly  acid  reaction  and  heat  again  :  no  coagulation 
occurs. 

Exercise  III. — Prepare  solutions  of  serum-albumin,  serum- 
globulin,  casein,  egg-albumin,  and  test  the  behavior  of  these  solu- 
tions to  sodium  chloride,  magnesium  sulphate,  and  ammonium 
sulphate  by  salting  the  solutions  to  saturation  ;  let  the  precijntates 
stand  over  night  and  examine  the  filtrates  the  next  day  ;  test  with 
the  biuret  test  (page  36)  to  ascertain  whether  the  precipitation  is 
complete. 

Determine  the  upper  and  lower  limits  of  precipitation  of  the 
different  albumins  in  neutral  solutions  w'ith  a  saturated  neutral 
aqueous  solution  of  ammonium  sulphate,  as  described  (page  33). 

Exercise  IV. — Prepare  dilute  aqueous  solutions  of  serum-albumin, 

473 


474  APPENDIX. 

egg-albumin,  and  albiimoses  (Witte's  ])eptone),  also  a  dilute  saline 
solution  of  serum-globulin,  and  perform  the  various  qualitative 
tests  outlined  on  page  35.  Kepeat  the  tests  with  an  albuminous 
urine.  Kemove  all  albumins  from  one  of  the  albuminous  solutions 
with  ferric  chloride  as  described,  filter,  and  demonstrate  the  absence 
of  albumin  in  the  filtrate  with  potassium  ferrocyanide  and  acetic 
acid. 

Exercise  V. — Perform  the  albumin  color-tests  w^ith  various 
albumins  (egg-albumin,  serum-albumin,  casein)  either  in  solution  or 
in  substance,  as  directed  on  page  36.  Note  the  negative  Molisch 
reaction  in  the  case  of  casein,  and  the  relative  amount  of  sulphur 
in  serum-albumin  as  compared  with  casein. 

If  no  glyoxylic  acid  is  available,  use  glacial  acetic  acid  in  the 
Adamkiewicz  reaction ;  if  the  reaction  is  negative,  add  a  small 
amount  of  a  solution  of  oxalic  acid  which  has  been  previously 
treated  with  sodium  amalgam. 

Exercise  VI. — Examine  solutions  of  different  albumins  with  the 
polarimeter ;  note  the  degree  of  laevorotation. 

Exercise  VII. — Place  100  grammes  of  dry  egg-albumin  in  a 
flask  holding  about  1  liter ;  add  500  c.c.  of  concentrated  hydro- 
chloric acid  and  boil  for  twelve  hours.  Let  cool ;  add  strong  caustic 
alkali  solution  until  the  reaction  is  feebly  acid ;  boil  with  much 
animal  charcoal  for  about  one-half  hour  and  filter.  In  the  filtrate 
look  for  Icucin,  tyrosin,  glutaminic  acid,  asparaginic  acid,  etc.,  as 
descril)ed  in  Exercise  XXIII. 

Exercise  VIII. — See  exercises  on  serum-albumin  and  serum- 
globulin.  Exercise  XLVI. 

Exercise  IX. — Suspend  a  small  amount  of  casein  (0.5  gramme) 
in  25  c.c.  of  water  and  dissolve  it  by  the  careful  addition  of  dilute 
caustic  alkali  solution  (1  :  10),  then  precipitate  the  casein  with  acetic 
acid  and  wash  with  water ;  this  is  to  remove  any  adherent  phos- 
phates. Now  fuse  the  purified  and  dried  casein  with  soda  and 
saltpeter  and  test  for  alkali  phosphate. 

Exercise  X. — See  Exercise  XXXVI. 

Exercise  XI. — Make  a  little  starch  paste ;  boil  with  25  per 
cent,  hydrochloric  acid  for  ten  minutes ;  filter ;  render  the  filtrate 
strongly  alkaline  with  caustic  soda  solution  and  test  for  glucose. 
(See  Eiercise  XXXYI.) 

Exercise  XII. — Prepare  a  90  per  cent,  solution  of  alcohol 
(100  c.c),  add  25  c.c.  of  a  concentrated  solution  of  caustic  soda, 
melt  about  50  grammes  of  lard,  and  pour  all  into  a  half  liter  flask. 
Heat  on  a  water-bath  and  shake  the  mixture  frequently  as  soon  as 
the  alcohol  begins  to  ))oil.  When  saponification  is  complete,  which 
occurs  very  rapidly  if  the  experiment  is  conducted  as  just  outlined, 
a  small  amount  of  the  solution  on  being  poured  into  distilled  water 
should  give  rise  to  no  turbidity.  The  resulting  solution  contains 
soap  and  glycerin,  which  can  be  demonstrated  according  to  the  usual 
methods. 


LABORATORY  EXERCISES.  475 

Exercise  XIII. — 1.  Collect  about  50  c.c.  of  saliva  by  chewing 
some  paraffin  or  rubber  and  spitting  into  a  beaker.  Note  the 
appearance  of  the  secretion  and  test  the  reaction. 

2.  Demonstrate  the  presence  of  ptyalin  as  described  on  page  123. 

3.  Demonstrate  the  salivary  mucin  (page  125). 

4.  Demonstrate  the  presence  of  sulphocyanides  (page  125). 

5.  Demonstrate  the  presence  of  nitrites  (page  125). 

Exercise  XIV. — Procure  gastric  juice  from  a  human  being  at 
the  height  of  digestion  ;  or  prepare  an  artificial  mixture  containing 
2  to  3  pro  mille  of  hydrochloric  acid,  some  essence  of  pepsin,  and 
albumoses  (liquid  peptonoids  or  panopeptone). 

1.  Test  the  reaction  with  litmus;  with  congo  red  (using  congo 
red  paper  or  a  very  dilute  solution). 

2.  Perform  Giinzburg's  test,  Topfer's  test,  and  Boas'  test  for  free 
hydrochloric  acid  (pages  131  and  132). 

3.  Determine  the  total  acidity  (page  128). 

4.  Determine  the  amount  of  free  and  combined  hydrochloric 
acid :  (a)  according  to  Topfer;  (h)  according  to  Morner  and  Sjoqvist; 
(c)  according  to  Leo. 

Exercise  XV. —  1.  With  the  artificial  gastric  juice  used  in  Exer- 
cise XIV.  test:  (a)  for  pepsin;  {h)  for  chymosin  (pages  139,  144). 
2.  Estimate  the  amount  of  pepsin  (page  142). 
Exercise  XVI. — Prepare  a  3  pro  mille  solution  of  lactic  acid. 

1.  Perform  Uftelmann's  test  and  Kelling's  test  (page  134). 

2.  Estimate  the  amount  according  to  Boas'  method  (page  135). 

3.  With  a  3  pro  mille  solution  of  acetic  acid  and  butyric  acid 
perform  the  tests  described  on  page  135. 

Exercise  XVII. — 1.  Prepare  a  solution  of  pancreatin  (0.5  per 
cent.)  in  a  0.25  to  1  per  cent,  solution  of  sodium  carbonate ;  add  a 
small  flake  of  fibrin  and  place  in  an  incubator  at  40°  C. 

2.  Prepare  a  neutral  aqueous  solution  of  pancreatin  (10-15  c.c.) 
and  add  a  small  flake  of  fibrin  ;  ])lace  in  an  incubator. 

3.  Prepare  an  acid  solution  of  pancreatin  in  a  similar  manner 
(containing  2  to  3  pro  mille  of  hydrochloric  acid),  add  a  small  flake 
of  fibrin,  and  place  in  an  incubator.  Observe  the  eflfect  at  the  end 
of  one  hour. 

4.  Perform  similar  tests  with  pepsin. 

5.  Note  that  previous  boiling  will  destroy  the  action  of  the  ferment. 
Exercise  XVIII. — From  a  10  per  cent,  solution  of  Witte's  peptone 

(1000  c.c.)  isolate  the  various  albumose  fractions  as  in  Exercise  III. 
Demonstrate  the  absence  of  a  carbohydrate  group  in  proto-albu- 
mose  and  hetero-albumose  ;  its  presence  in  the  deutero-fraction  B. 
Isolate  gluco-albumose  from  the  B-fraction,  and  also  thio-albumose 
(test  the  sulphur  reaction  in  this,  page  39). 

Exercise  XIX. — 1.  Procure  250  c.c.  of  ox-bile  and  of  sheep-bile. 
Note  the  general  characteristics  of  each. 

2.  From  the  ox-bile  prepare  Platner's  bile,  and  isolate  from  it 
glycocholic  acid  and  taurocholic  acid  (page  162). 


476  APPENDTX. 

3.  With  Platner's  bile  and  the  isolated  acids  perform  Petten- 
kofer's  test  (page  103). 

4.  Isolate  taurin  from  the  sheep-bile  and  identify  it  (page  171). 

5.  With  the  fresh  sheep-bile,  somewhat  diluted,  perform  Gmelin's 
test,  Hu])pert's  test,  and  Smith's  test  (page  175). 

6.  Isolate  the  biliary  mucin  from  ox-bile  (page  161). 

7.  Procure  some  human  bile  (at  autopsy)  and  examine  into  the 
presence  of  true  mucin  (page  161). 

8.  Procure  some  gall-stones  (preferably  cholesterin  stones) ;  isolate 
the  cholesterin  in  pure  form  and  perform  the  Liebermann-Burck- 
hard  and  Salkowski  tests  (page  180). 

Exercise  XX.— Carbohydrate  digestion  (see  Exercise  XIII.). 

Exercise  XXI. — 1.  Digest  100  grammes  of  fresh  fibrin  with 
pepsin-hydrochloric  acid  and  isolate  the  various  fractions  as  de- 
scribed on  page  198.  If  fibrin  preserved  in  chloroform  is  only  avail- 
able, heat  this  in  a  2  per  cent,  solution  of  hydrochloric  acid  until  it 
swells  and  assumes  a  jelly-like  appearance. 

Exercise  XXII. — Digest  100  grammes  of  fibrin  with  pancreatin 
and  isolate  tiie  various  albumoses  as  described  on  page  199. 

Exercise  XXIII. — From  a  fibrin-pancreatin  digestive  mixture 
that  has  stood  for  a  week  well  guarded  against  putrefactive  changes 
with  toluol  (2  cm.  of  toluol  should  stand  above  the  mixture),  isolate 
(a)  the  anti-peptone  fraction.  Demonstrate  that  this  contains 
a  fraction  which  can  be  precipitated  with  phosphotungstic  acid 
and  one  which  remains  in  solution  (page  204).  (6)  Use  this  same 
mixture  to  demonstrate  the  presence  of  leucin  and  tyrosin  (page  208), 
asparaginic  acid  and  glutaminic  acid  (page  209).  (c)  In  the  same 
mixture  demonstrate  the  presence  of  tryptophan  (page  212). 

4.  To  demonstrate  glycocoll  as  a  decomposition-product  of  the 
albumins  it  is  best  to  start  with  gelatin.  This  is  conveniently 
hydrolyzed  with  25  per  cent,  sulphuric  acid  (page  210).  Use  about 
100  grammes  of  gelatin,  1000  c.c.  of  dilute  acid,  and  boil  for  twenty- 
four  hours. 

Exercise  XXIV. — Hash  100  grammes  of  liver-tissue  ;  suspend  in 
1000  c.c.  of  water ;  add  an  abundance  of  toluol,  shake  well  (the  toluol 
should  form  a  layer  1  cm.  deepen  the  surface),  and  keep  at  a  tem- 
perature of  40°  C.  for  three  to  four  weeks.  Then  examine  for 
albumoses,  tyrosin,  leucin,  etc.,  as  above. 

Exercise  XXV. — Hash  100  grammes  of  pancreas  ;  suspend  in 
1000  c.c.  of  water ;  place  in  an  incubator  at  40°  C.  for  a  week,  and 
then  examine  for  indol,  skatol,  and  phenol  (page  224). 

Exercise  XXVI. — 1.  Collect  the  urine  of  twenty-four  hours 
from  an  individual.  Measure  the  amount,  note  the  color,  odor,  reac- 
tion, and  specific  gravity  (page  227). 

2.  Determine  the  degree  of  acidity  according  to  Freund's  method 
(page  233). 

3.  Estimate  the  mineral  ash  (page  237). 

Exercise  XXVII. — Estimate  the  chlorides  of  the  urine  (use  the 


LABORATORY  EXERCISES.  477 

collected  amount  of  twenty-four  hours  in  all  the  quantitative  work 
on  tlie  urine  which  is  to  follow)  (page  237). 

2.  Estimate  the  phosphates  :  (a)  total ;  (6)  the  earthy  ;  and  (c)  the 
alkaline  (page  238). 

3.  Estimate  :  (a)  the  total  sulphates  ;  (6)  the  conjugate  sulphates  ; 
(c)  the  neutral  sulphur  (pages  238,  296). 

Exercise  XXVIII. — 1.  Prepare  some  urea  from  the  urine  (page 
247) ;  examine  with  a  microscope  the  urea  nitrate  which  is  formed. 

2.  Dissolve  a  few  crystals  of  urea  in  1  c.c.  of  water  and  add  a 
small  amount  of  a  strong  solution  of  oxalic  acid  ;  urea  oxalate  is 
precipitated  ;  examine  with  a  microscope. 

3.  Heat  some  urea  in  a  test-tube  as  described  on  page  247  ;  note 
the  formation  of  biuret  (page  37). 

4.  Estimate  the  urea  with  sodium  hypobromite  solution,  using 
Doremus'  ureometer ;  the  gas  evolved  is  nitrogen;  the  CO^  which 
is  formed  is  taken  up  by  the  excess  of  sodium  hydrate  in  the  hypo- 
bromite solution  (page  246). 

5.  Estimate  the  urea  according  to  Folin's  method  (page  249). 
Exercise  XXIX. — Determine  the  total  amount  of  nitrogen  in  the 

urine  according  to  Kjeldahl's  method  (page  250). 

Exercise  XXX. — Isolate  some  uric  acid  from  human  urine 
(page  256). 

2.  Note  the  insolubility  in  water. 

3.  Perform  the  raurexid  test,  the  copper  test,  Dennige's  test,  and 
Schiff's  test  (page  256). 

4.  Estimate  the  uric  acid  according  to  Folin's  method  (page  257). 
Exercise  XXXI. — 1.   Estimate  the  oxalic  acid  according  to  Dun- 
lop's  method  (page  261). 

2.  Isolate  kreatinin  as  kreatinin-zinc  chloride  (page  265). 

Exercise  XXXII. — 1.  Test  the  urine  for  indican,  using  Jaffe's 
test,  or  Obermeyer's  test  (page  271). 

2.  Estimate  the  indican  according  to  Wang's  method  (page  272). 

Exercise  XXXIII. — Feed  a  dog  a  couple  of  grammes  of  chloral 
and  demonstrate  glucuronates  in  the  urine  the  next  day  (page  276). 

Exercise  XXXIV. — Prepare  artificial  diabetic  urine,  containing 
/9-oxybutyric  acid,  diacetic  acid,  acetone,  and  glucose ;  let  stand  for 
several  hours. 

1.  Polarize  the  urine  and  note  the  degree  of  dextrorotation. 

2.  Ferment  the  urine  with  yeast  for  twenty-four  hours  and  re- 
examine (la3vorotation  due  to  oxybutyric  acid). 

3.  Estimate  the  oxybutyric  acid  as  a  a-crotonic  acid  (page  286). 

4.  Test  for  diacetic  acid,  using  both  Arnold's  and  Gerhardt's  test, 
.(pages  287  and  288). 

5.  Test  for  acetone  in  the  distillate  (see  page  289)  ;  use  Legal's, 
Lieben's,  and  Gunning's  tests  (pages  289). 

6.  Estimate  the  acetone  (page  290). 

Exercise  XXXV. — 1.  Prepare  some  cystin  from  human  hair  as 
•described  on  page  296. 


478  APPENDIX. 

2.  Examine  the  resulting  crystals  with  a  microscope  and  test 
their  solubility  in  ammonia,  caustic  soda,  and  hydrochloric  acid. 

3.  Prove  that  the  substance  contains  sulphur,  as  follows  :  place  a 
small  amount  in  a  test-tube,  dissolve  in  caustic  alkali,  add  a  few 
drops  of  acetate  of  lead  solution,  and  boil;  note  the  brownish-black 
color  which  results  owing  to  the  formation  of  sulphide  of  load. 

4.  Repeat  the  preceding  experiment :  (a)  with  normal  urine ; 
(l>)  with  urine  to  which  a  solution  of  cystin  has  been  added. 

5.  Estimate  the  amount  of  neutral  sulphur  in  the  urine  as  de- 
scribed on  page  296.     (Be  careful  with  the  sodium  peroxide.) 

Exercise  XXXVI. — 1.  Prepare  a  solution  of  glucose  in  water 
(al)out  1  per  cent.)  or  in  urine,  and  perform  :  («)  Xylander's  test ; 
(b)  Fehling's  test ;  (e)  the  fermentation  test ;  and  [d)  the  phenyl- 
hydrazin  test,  as  described  on  |)ages  299  and  300. 

2.  Determine  the  melting-point  of:  («)  phenyl-glucosazon  ;  to  this 
end,  purify  the  crystals  and  dry  them  as  described  on  page  301. 
Prepare  a  capillary  tube  about  Ih  inches  long  and  with  a  lumen  of 
approximately  1  mm.,  seal  one  end,  place  some  ciystals  of  the  osazon 
in  the  interior ;  fasten  the  tube  with  platinum  wire  to  the  bulb  of  a 
thermometer  registering  about  250°  C. ;  suspend  the  thermometer  in 
concentrated  sulphuric  acid  in  a  beaker;  heat  gently  and  note  the 
temperature  at  which  the  crystals  melt.  In  a  similar  manner  prepare 
the  osazons  of :  (6)  lactose,  (e)  Isevulose,  and  {(I)  maltose,  and  deter- 
mine the  melting-points  as  described. 

3.  Estimate  the  amount  of  glucose  in  solution  :  («)  with  the 
polarimeter  ;  (6)  by  the  diiierential  density  method  ;  (c)  by  Knapp's 
method  ;  and  {d)  bv  Fehling's  method.  (The  solution  of  glucose 
should  stand  a  few  hours  before  being  polarized.) 

Exercise  XXXVII. — 1.  Prepare  a  dilute  solution  of  arabinose 
(0.1  percent.);  perform  the  orcin  test  as  described  by  Bial  (page  306). 

2.  Test  with  Fehling's  solution. 

3.  Prepare  the  corresponding  osazon  and  determine  the  melting- 
point  (fice  preceding  Exercise). 

Exercise  XXXVIII. — 1.  Procure  some  albuminous  urine  and 
perform  :  ((()  the  nitric  acid  test ;  {b)  the  boiling-test  ;  (c)  the  potas- 
sium ferrocyanide  test  (pages  308  to  310). 

2.  Demonstrate  the  presence  of  two  albumins  in  such  urine,  one 
of  which  coagulates  betAveen  50°  and  60°  C,  and  the  other  between 
65°  and  75°  C. ;  note  that  the  former  is  present  in  smaller  amount, 
^lake  the  examination  by  hanging  a  ihermometer  registering  100°  C 
in  a  wide  test-tube  about  one-third  full  of  urine  ;  immerse  this  well 
in  a  large  beaker  of  water  ;  heat  very  slowly.  Prove  that  one  of  the 
two  albumins  is  serum-albumin  and  the  other  serum -globulin 
(page  310). 

3.  Estimate  the  amount  of  albumin  :  (a)  graviraetrically  ;  (6)  by 
Esbach's  method. 

4.  Procure  urine  from  a  case  of  acute  cystitis.  Test  for  nucleo- 
albumin  as  described  on  page  310. 


LABORATORY  EXERCISES.  479 

5.  Add  a  couple  of  srrammes  of  AVitte-peptone  to  a  liter  of  urine ; 
then  examine  the  solution  as  described  on  page  311. 

Exercise  XXXIX. — 1.  Procure  some  febrile  urine  and  examine 
this  :  (aj  for  urochrome  ;  (b)  for  uroerythrin  ;  (e)  for  urobilin. 

2.  Procure  some  typhoid  urine  between  the  seventh  and  the  tenth 
day  of  the  disease  and  apply  Ehrlich's  test.  Use  about  5  c.c.  of 
urine  and  an  equal  amount  of  the  sulj)hanilic  acid  mixture.  After 
adding  the  ammonia  to  fjrm  a  layer  above  the  urine  and  acid  mix- 
ture note  the  color-ring  at  the  zone  of  contact.  Shake  the  fluid  and 
note  the  color  of  the  foam  ;  pour  all  into  a  white  basin  and  dilute 
copiously  with  water ;  note  the  salmon  pink.  Repeat  the  experi- 
ment with  normal  urine. 

3.  Add  a  little  blood  to  urine  :  («)  perform  Heller's  test ;  [h) 
examine  the  urine  spectroscopically  (page  317). 

4.  Procure  some  urine  containing  hrematoporphyrin  :  («)  examine 
this  spectroscopically  :  (b)  isolate  the  htematoporphyrin  and  examine 
its  pure  solution  with  a  spectroscope  (page  318). 

5.  With  urine  from  a  case  of  melanotic  sarcoma  perform  :  (a) 
the  bromine  test ;  (b)  the  iron  test  (page  319). 

6.  Procure  bile  from  a  dog  or  from  a  himian  being  (at  autopsy"), 
dilute  with  water,  and  perform  the  various  tests  described  on 
page  175. 

Exercise  XL. — 1.  Procure  blood  at  a  slaughter-house;  let  a  por- 
tion flow  directly  from  the  vessel  into  a  saturated  sokition  of  sodium 
sulphate  or  a  10  per  cent,  solution  of  sodium  chloride  (equal  parts) ; 
receive  a  second  portion  in  a  dry  cylinder.  Note  that  in  the  first 
portion  coagulation  does  not  occur.  Observe  the  phenomenon  of 
coagidation  in  the  second.  Prepare  also  some  oxalate  plasma 
and  some  albumose  plasma  (page  336).  Keep  some  oxalate  plasma 
on  ice  and  note  the  separation  of  the  corpuscles  on  standing. 

2.  Determine  the  specific  gravity  of  human  blood  according  to 
Hammerschlag's  method  (page  331). 

3.  Determine  the  reaction  of  fresh  cat's  or  dog's  blood  according 
to  Lowy's  method,  and  that  of  human  blood  according  to  Dare 
(page  333). 

4.  Isolate  the  fibrinogen  from  ai')out  50  c.c.  of  plasma  bv  half- 
saturation  with  sodium  chloride  (page  337).  To  the  resulting  solu- 
tion of  fibrinogen  add  a  little  serum.  Note  the  occurrence  of 
coagulation. 

5.  Prepare  blood-serum  by  allowing  defibrinated  blood  (500  c.c.) 
to  stand  on  ice,  or,  still  better,  by  separating  the  cellular  elements 
by  centrifugation. 

6.  From  100  c.c.  of  serum  isolate  the  serum-albumin  fraction 
and  the  globulin  fraction  bv  means  of  ammonium  sulphate  (pages 
339  and  340). 

A.  Note  the  behavior  of  the  globulin  fraction  (a)  on  dialysis 
(examine  the  remaining  solution  for  globulins  by  saturation  with 
magnesium   sulphate),  (6)  on  passing  a  stream  of  CO2  through  a 


480  APPENDIX. 

dilute  solution  of  the  globulin,  (c)  on  diluting  20  times  with  water 
after  acidifying  faintly  with  acetic  acid  (page  339).  Xote  the  coag- 
ulation-point of  the  serum-globulin  in  the  presence  of  5  per  cent, 
of  sodium  chloride  as  described  below. 

B.  Note  the  solubility  of  the  serum-albumin  in  water ;  estimate 
the  coagulation-point  in  the  presence  of  5  per  cent,  of  sodium 
cidoride.  To  this  end,  a  test-tube  is  filled  about  one-half  with  the 
solution  and  immersed  in  a  large  beaker  with  water;  a  thermometer 
registering  100°  C.  should  dip  nearly  to  the  bottom  of  the  test-tube; 
heat  slowly. 

7.  Remove  all  albumins  from  20  to  30  c.c.  of  serum,  according 
to  Cavazzani's  method  (page  340).  In  the  resulting  solution  de- 
monstrate the  presence  of  glucose  by  means  of  the  phenyl-hydrazin 
test. 

Exercise  XLI. — 1.  Examine  fresh  blood  directly  with  a  spectro- 
scope. 

2.  Estimate  the  amount  of  haemoglobin  in  human  blood  with 
Dare's  hsemoglobinometer,  or  with  v,  Fleischl's  hoemometer. 

3.  Prepare  some  crystalline  oxyhtemoglobin  from  dog's  blood 
(100  c.c.)  (page  357)  ;  examine  the  resulting  product  spectroscopi- 
cally  ;  note  the  change  in  the  spectrum  on  the  addition  of  caustic 
alkali  and  reduction  with  ammonium  sul{)hide  (page  354). 

4.  Prepare  some  hsemin  from  oxy haemoglobin,  and  from  this  in 
turn  hsematin  (page  356);  note  the  spectrum  of  the  latter  in  acid 
and  in  alkaline  solution. 

5.  Perform  the  hremin  test  with  a  drop  of  blood.  To  this  end, 
let  a  delicate  film  of  sodium  chloride  form  in  the  middle  of  a  slide ; 
place  on  this  a  drop  of  blood  ;  let  dry  and  add  glacial  acetic  acid,  a 
drop  at  a  time,  while  heating  very  gently  over  a  flame;  examine 
from  time  to  time  with  a  microscope.  Finally  add  a  di'op  of 
glycerin,  cover,  and  examine.  Note  the  color,  form,  and  varying 
size  of  the  crystals. 

6.  Prepare  some  carbon  monoxide  hremoglobin  from  oxyhjemo- 
globin  (page  360).     Note  the  spectrum. 

7.  Prepare  a  solution  of  hitmatoporphyrin  in  a  test-tube  by 
adding  about  5  drops  of  blood,  drop  by  drop,  and  shaking  con- 
stantly, to  8  or  10  c.c.  of  concentrated  sulphuric  acid  ;  examine  with 
a  spectroscope  (page  363). 

Exercise  XLII. — 1.  Hash  100  grammes  of  lean  muscle-tissue 
(beef);  suspend  in  300  c.c.  of  water;  stir  well  ;  allow  to  stand  for 
a  couple  of  hours  ;  filter  through  muslin  and  then  through  filter- 
paper,  [a)  Examine  the  reaction  of  the  filtrate.  (Jj)  Demonstrate 
the  presence  of  three  albumin  fractions  by  fractional  coagulation. 
(Arrange  the  apparatus  as  described  above,  XL.,  6,  B.)  Heat  very 
slowly  and  filter  whenever  one  fraction  has  been  coagulated,  (c) 
Prepare  mvosin  from  the  meat  residue  after  thorough  washing  with 
Avater  as  follows:  prepare  a  15  per  cent,  solution  of  ammonium 
chloride,  suspend  the  meat  in  this ;  stir  mcU  and  allow  to  stand  for 


LABORATORY  EXERCISES.  481 

twentv-four  hours.  The  resulting  solution  contains  the  myosin. 
Note  the  readiness  witii  which  myosin  coagulates  by  dilating  a  few 
CO.  of  the  solution  with  water,  and  by  adding  finely  powdered  salt 
lo  the  solution  and  stirring.  Boil  a  few  c.c.  of  the  solution,  filter, 
and  examine  the  filtrate  for  calcium  salts  by  adding  ammonium 
oxalate. 

2.  Prepare  muscle-plasma  and  isolate  the  myogen  from  this  as 
described  on  page  374.     Examine  the  resulting  product. 

3.  Demonstrate  the  presence  of  glycogen  in  fresh  muscle-tissue 
(page  382). 

4.  Demonstrate  the  presence  of  glucose  (page  38.")). 

5.  Demonstrate  the  presence  of  lactic  acid  (using  about  100 
grammes  of  meat)  (page  386). 

6.  Demonstrate  tlie  presence  of  kreatin  (page  390). 

7.  To  demonstrate  the  xanthin  bases  it  is  most  convenient  to  start 
with  a  meat  extract,  for  example,  with  100  grammes  of  Liebig's 
extract.     Proceed  with  this  as  described  on  page  391. 

Exercise  XLIII. — 1.  Isolate  protagon  from  a  fresh  calf's  brain 
(page  401). 

2.  Demonstrate  the  presence  of  lethicins  in  the  brain  (page  403). 

Exercise  XLIV. — 1.  Decalcify  pieces  of  bone  with  50  per  cent, 
hydrochloric  acid  ;  let  stand  from  twenty-four  to  forty-eight  hours. 
Decant  the  acid  solution  which  contains  the  mineral  constituents. 
Wash  the  remaining  ossein  with  water  and  with  dilute  soda  solution, 
then  place  in  a  bowl  with  water  and  boil  until  the  ossein  has  dis- 
solved, neutralize  or  make  faintly  alkaline  with  soda,  decant,  and 
cool;  note  that  the  solution  jellies  (gelatin). 

2.  Froui  cartilage-shavings  isolate  the  chondromucoid  (page  414). 

Exercise  XLV. — 1.  Demonstrate  the  presence  of  iron-containing 
albuminates  in  the  liver  (page  433).  Use  about  100  grammes  of 
tissue. 

2,  Feed  a  rabbit  about  25  grammes  of  glucose  through  a  tube  ; 
kill  it  several  hours  later,  and  demonstrate  the  presence  of  glycogen 
in  the  liver  (page  435) ;  use  a  portion  of  the  same  liver  for  the 
following  experiment : 

3.  The  demonstration  of  glucose  (page  435). 

Exercise  XL VI. — Prepare  guanylic  acid  from  pancreas  as  de- 
scribed on  page  436. 

Exercise  XL VII. — 1.  Xote  the  general  physical  properties  of 
milk;  examine  a  drop  microscopically;  note  the  reaction  and  the 
specific  gravity  ;  boil  some  milk. 

2.  Isolate  the  d liferent  albumins  of  the  milk  (page  446). 

3.  Estimate  the  total  albumins  (page  446). 

4.  Add  some  essence  of  pepsin  (rennin)  to  milk  ;  place  in  the 
incubator  at  40°  C.  and  note  the  occurrence  of  coagulation  (page 
444). 

5.  Let  a  specimen  of  milk  stand  exposed  to  the  air  for  forty-eight 
hours  ;  note  the  reaction.     What  has  occurred  ?     (Page  445.) 

31 


482  APPENDIX. 

6.  Estimate  the  amount  of  fat  (page  448). 

7.  Estimate  the  amount  of  hictose  (page  448). 

8.  Perform  the  test  of  Umikoff  for  citric  acid  (page  449). 
Exercise  XL VIII. — 1.   Demonstrate  the  presence  of  the  various 

albumins  in   wliite  of  egg  (use  the  whites  of  about  20  eggs)  (page 
458). 

2.  Isolate  the  ovomucoid  (page  459). 

3.  Demonstrate  the  lecithins  in  the  yolk  (page  463). 


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1,  Absorption-spectrum  of  a  solution  of  oxyhrcmodobin ;  2,  of  a  solution  of  hremoglobin ;  3, 
of  a  feebly  alkaline  solution  of  methrr-moRlobin  ;  4,  of  a  solution  of  hrematin  in  acid  ether 
(oxalic  acid) ;  5,  of  an  alkaline  solution  of  hrematin  :  6,  of  an  alkaline  solution  of  hsemo- 
chromogen:  7,  of  an  acid  solution  of  urobilin;  8,  of  an  ammoniaeal  solution  of  urobilin, 
after  the  addition  of  zinc  chloride  solution;  9,  of  a  solution  of  lutein  (ethereal  extract 
of  the  volk  of  egg). 

483 


INDEX. 


ACCIPENSERIN,  54 
Acetic  acid,  91 

in  stomacli  contents,  135 
tests  for,  135 
Acetone,  92,  286,  288 

estimation  of,  290 

tests  for,  289 
Achroodextrin,  123,  183 
Acid,  acetic,  135 

adenylic,  96 

alloxyproteinic,  292 

arachinic,  447 

asparaginic,  208 

avivitellinic,  461 

barbituric,  105 

benzoic,  278 

bilianic,  168 

bilirubinic,  174 

biliverdinic,  175 

butyric,  91,  135 

capronic,  91 

carbamic,  241 

carnic,  377 

chenocholalic,  169 

chenotaurocholic,  166 

chitonic,  68 

cholalic,  167 

cholanic,  164,  169 

choieic,  169 

choleo-camphoric,  168 

cholesteric,  169 

choloidinic,  16S 

cholonic,  171 

chondroitin-sulphuric,  51,  275 

cilianic,  168 

citric,  449 

crotonic,  286 

cvsteinic,  170 

damalic,  283 

damaluric,  283 

debydrocholalic,  168 

debydrocboleic,  169 

desoxvcholalic,  169 

diacetic,  92,  286,  287 

dialuric,  105,  253 

dioxy-phenyl-acetic,  280 

ethylene-succinic,  82 

fatty,  91,  284 

fellic,  169 

ferri-albuniinic,  433 

formic,  91 

galactonic,  70 


Acid,  gluconic,  67,  275 
glucuronic,  67,  274 
glutaminic,  85,  209 
glutaric,  93 

glycerin-phosphoric,  78,  321 
glycocholic,  87,  164 
glycolic,  92 
glycoluric,  105 
glycosuric,  280 
glyoxylic,  263 
guanidin-butyric,  83 
guano-biliary,  165 
guanylic,  96,  436 
haematinic,  356,  363 
hippuric,  87,  276 
homogentisinic,  280 
hydantoic,  105 
hydrochloric,  129 
hydrocinnamic,  94 
hydroparacuniaric,  93,  279 
hydurilic,  105 
hyocholalic,  169 
hyoglycocholic,  165 
hyotaurocholic,  166 
ichthulinic,  462 
inosinic,  96,  394 
isobilianic,  168 
kynurenic,  283 
kvnuric,  283 
lactic,  92,  134,  290 
la?yulinic,  68,  99 
laurinic,  447 
leucinic,  92 
lithofellic,  169 
lithuric,  283 
lysuric,  84 
mannonic,  67 
manno-saccharinic,  67 
methyl-hydantoinic,  87 
mucin  ic,  67 
myristinic,  180,  447 
nucleinic,  57,  95 
oleic,  77 
ornithuric,  84 
oxalic,  93,  260 
oxaluric,  105,  261 
oxy-amygdalic,  280 
oxy-butyric,  285 
oxy-hydroparacumaric,  93 
oxy-proteinic,  292 
oxy-tricarballylic,  449 
palmitic,  77 

485 


486 


INDEX. 


Acid,  parabanic,  105,  107 

paralactif,  290 

para-oxy-beiizoic,  94 

para-oxy-plionyl-acetic,  88,  279 

para-oxy-phcnyl-glycolic,  280 

para-oxy-plieiiyl-lactic,  93,  279 

para-oxy-phenyl-propionic,    88,    94, 
279 

phenaceturic,  87,  95,  279 

phonyl-acetic,  94,  279 

phenyl-propionic,  94,  279 

plio.spho-carnic,  377 

pliyliocyanic,  21 

plasminic,  98 

propionic,  91 

pyrocatechuic,  280 

pyrocholesteric,  1C9 

rhodizonic,  387 

saccharinic,  67,  274 

saccharo-lactonic,  67 

sarcylic,  96 

skatol-oarbonic,  90,  217,  273 

sperma-nucleinic,  95 

stearic,  77 

tartronic,  106 

taurocarbaminic,  87,  293 

taurocholic,  87,  166 

tetraoxy-amido-capronic,  51 

thyniinic,  97 

thymo-nucleinic,  96 

trioxy-phenyl-propionic,  280 

tyrosin-hydantoinic,  87 

uramic,  87 

uramido-benzoic,  87 

uric,  101,  104,  105,  252 

urocaninic,  283 

urochloraJic,  275 

uroferric,  292 

uroleiicinic,  280 

uroproteic,  292 

uroxanlbinic,  280 

urrhodinic,  280 

ursocholeic,  171 

valerianic,  91 

xanthylic,  96 

yeast-nucleinic,  96 
Acids,  aromatic  oxy-,  279 

biliary,  161 
Acrolein,  78 

Adamkiewicz'  reaction,  38 
Addison's  disease,  469 
Adenin,  102,  393 

isolation  of,  391 

tests  for,  394 
Adenylic  acid,  96 
Adipocere,  421 
Adipose  tissue,  418 

analysis  of,  419,  420 
origin  of,  420 
significance  of,  422 
Adrenal  glands,  469 
Adrenalin,  Takamine's,  470 
Albamin,  45 


Albumen  of  birds'  eggs,  456 

albumins  of,  456 

analysis  of,  456 

ovalbumins  of,  457 

ovomucoid  of,  459 

tata,  456 
Albumin  of  Bence  Jones,  311 
Albuminates,  61 

alkaline,  61 

of  iron,  431 
Albuminoids,  59 

digestion  of,  192 
Albumins,  28,  149 

behavior  toward  alcohol,  35 
toward  ncuti-al  salts,  33 
toward  polarized  light,  30 

classification  of,  48 

coagulated,  61 

coagulation  of,  30 

color-reactions  of,  36 

crystallization  of,  29 

decomposition  of,  39 

denaturization  of,  32 

derived,  61 

difiusion  of,  30 

digestion  of,  185 

elementary  composition  of,  28 

estimation  of,  in  the  blood,  340 
in  milk,  446 
in  plasma,  316 
in  urine,  313 

molecular  size  of,  48 

native,  49 

nitrogenous  derivatives  of,  82 

of  the  albumen,  456 

of  the  blood,  336 

of  the  lymph,  367 

of  the  milk,  443 

of  the  muscle-tissue,  373 

of  the  nerve-tissue,  398 

of  the  urine,  307 

of  the  yolk,  461 

precipitation  of,  35 

reaction  of,  28 

solubility  of,  29 

special  reactions  of,  35 

structural  composition  of,  39 

synthesis  of,  26 

tests  for,  in  the  urine,  308 
Albuminuria,  307 

physiological,  307 
Albumoid,  415 

isolation  of,  415 
Albumose-plasma,  336 
Albumoses,  43,  62,  186 

analvsis  of,  200 

in  the  blood.  348 

in  ihe  urine,  311 

primary,  43,  62,  186 

secondary,  43,  62,  187,  201 

reactions' of,  200,  202,  203 

tests  for,  in  the  urine,  311 
Aldehydase,  119 


liiDEX. 


487 


Aldoses,  66 
Aleiiron  crystals,  "29 
Alkakiidal  reagents,  36 
Alkapton,  280 " 
Allantoic  acid,  105 
Allantoin,  107,  370,^  465 

in  the  urine,  262 

isolation  of,  263 
Allantnric  acid,  105 
Allituric  acid,  105 
Alloxan,  104,  107  _ 
Alloxanic  acid,  105 
Alloxantin,  105 
Alloxiiric  bases,  101 
AUoxyproteinic  acid,  292 
Aniiilo-acids,  82 
Ainido-nitrogen,  47 
Aniidf)-thio-]actic  acid,  88 
Amididin,  183 

Auinionia,  estimation  of,  in  the  urine,  250 
Amnioniinii  piirpurate,  107 
Amniotic  fluid,  analysis  of,  370 
Amphikreatin,  388 
Amphikreatinin,  108 
Amphopeptone,  63,  186 
Amygdalin,  25 
Amylases,  117 
Amylodextrin,  73 
Amyloid,  60 
Amvlolytic  ferment  of  pancreatic  juice, 

lo2 
Amylopsin,  152 
Amvlum,  73 
Animal  cell,  324 

fat,  418 

gum,  50 

sinistrin,  50 
Anti-alburaid,  188 
Antipeptone,  63,  189,  204 

preparation  of,  204,  213 
Aqueous  humor,  analysis  of,  369,  406 
Arabinose,  70,  306 
Arachinic  acid,  447 
Arbacin,  53 
Arbutin,  25 
Arginin,  40,  48,  82 

in  the  spleen,  438 
Arnold's  test  for  diacetic  acid,  287 
Aromatic  constituents  of  the  urine,  279 

oxy-acids,  93 

in  the  urine,  279 
Arterin,  351 

Ascherson's  haptogenic  membrane,  439 
Asparagin,  209 
Asparaginic  acid,  40,  208 
isolation  of,  209 
Autolysis,  114,  197 
Avivitellinic  acid,  461 

BACTERIAL  action  in  the  intestinal 
tract,  214 
decomposition    of    biliary    constitu- 
ents, 220 


Bacterial  decomposition  of  fats,  219 

Barbituric  acid,  105 

Bayer's  indigo-purpurin,  270 

Bence  Jones'  albunun,  311 

Benzoic  acid,  isolation  of,  from  hippuric 

acid,  278 
Bezaar  stones,  169 
Bile,  157 

acids  of  (see  also  Biliary  acids),  161 

amount  of,  159 

chemical  composition  of,  159 

crystallized,  of  Platner,  162 

general  properties  of,  159 

mucinous  body  of,  161 

pigments  of,  172 

in  the  urine,  320 

secretion  of,  158 

significance  of,  157 
Bilianic  acid,  168 
Biliary  acids,  161 

formation  of,  161 
in  the  urine,  320 
isolation  of,  162 
tests  for,  163 

constituents,  bacterial  decompositiou 
of,  220 

iron,  181 
Bilicyanin,  178 
Bilifuscin,  178 
Bilihumin,  179 
Biliprasin,  175,  178 
Biliimrpurin,  178 
Bilirubin,  173 

isolation  of,  176 

tests  for,  175 
Bilirubinic  acid,  173 
Biliverdin,  177 

isolation  of,  177 
Biliverdinic  acid,  175 
Bilixanthin,  178 
Biuret,  37,  100 

reaction,  36 
Blood,  329 

amount  of,  332 

chemical  composition  of,  334 
examination  of,  332 

coagulation  of,  342 

color  of,  330  , 

extractives  of,  348 

fat  in,  348 

fibrin-ferment  in,  342 

fibrinogen  in,  337 

glucose  in,  318 

glycogen  in,  348 

leucocytes  of,  329 

odor  of,  331 

physical  characteristics  of,  330 

pigments  of,  352 

plaques  of,  329 

reaction  of,  332 

red  corpuscles  of,  329,  351 

serum-albumin  of,  339 

serum-globulin  of,  337 


488 


ISDEX. 


Blood,  specific  gravity  of,  331 

taste  of,  331 

urea  in,  348 
Blood-pigments  in  the  mine,  317 
Blood-plasma,  330,  336 
Blood-seium,  330,  341 
Boas'  method  of  estimating  lactic  acid, 
135 

test  for  hydrochloric  acid,  132 
for  lactic  acid,  134 
Boiling  test  for  albumin,  309 . 
Bone,  415 

analysis  of,  416 
Bone-marrow,  417 
Bottcher's  crystals,  453 
Brain-tissue,  analysis  of,  397 
Brunner's  glands,  secretion  of,  153 
Bufidin,  429 
Bunge's  fluid,  433 
Butter,  440 
Buttermilk,  440,  443 
Butyric  acid,  91 

in  stomach  contents,  135 
tests  for,  135 

CACHEXIA  strumipriva,  468 
Cadaverin,  84 

in  the  urine,  322 
Cafiein,  102 

Calcium-thrombosin,  343 
Cane-sugar,  71 
Capronic  acid,  91 
Carbamic  acid,  241 
Carbohsemoglobin,  360 
Carbohydrates,  65 

fermentation  of,  68 

of  the  urine,  297 

synthesis  of,  in  plants,  23 
Carbon  dioxide  haemoglobin,  360 
methsemoglobin,  362 

monoxide  haemoglobin,  360 
Carnic  acid,  377 
Carniferrin,  377 
Carnin,  103 

in  muscle-tissue,  394 

isolation  of,  391 

properties  of,  394 

tests  for,  394 
Camosin,  395 
Cartilage,  412 

albumoid  in,  415 

analysis  of,  413 

chondroitin-sulphuric  acid  in,  413 

chondronuicoid  in,  414 

embryonic,  412 

mineral  siilts  in,  415 
Casein,  56,  443 

digestion  of,  191 

estimation  of,  446 

isolation  of,  446 

origin  of,  444 

properties  of,  443 
Caseoses,  62 


Castoreum,  429 

Cavazzani's  method  to  remove  albumins 

from  the  blood,  340 
Cell-globulins,  338 
Celluloses,  74 

Cement-substance  of  teeth,  417 
Cerebrin,  401 

isolation  of,  402 

properties  of,  401 
Cerebrosides  in  nerve-tissue,  400 
Cerebrospinal  fluid,  analysis  oi,  370 
Cerumen,  429 
Cetin,  77 
Cetylid,  402 

Charcol-Levden  crystals,  453 
Cheese,  440,  444 
Chenocholalic  acid,  169 
Chenotaurocholic  acid,  166 
Chitin,  418 
Chitonic  acid,  68 
Chitosamin,  50 
Chitose,  68 
Chlorides,   estimation   of,  in   the   urine, 

237 
Chlorophane,  410 
Chlorophyl,  20 

chemical  nature  of,  21 

granules,  20 

hydride,  24 
Cholalic  acid,  167 

isolation  of,  169 
Cholanic  acid,  169 
Cholecyanin,  178 
Choleglobins,  181 
Choleic  acid,  169 
Choleocamphoric  acid,  168 
Cholestearic  acid,  169 
Cholesterilins,  179 
Cholesterin,  80 

in  nerve-tissue,  404 

in  the  bile,  179 

in  the  urine,  320 

isolation  of,  180 

tests  for,  180 
Choletelin,  178,  313 
Cholin,  79 

Choloidinic  acid,  168 
Cholonic  acid,  164,  171 
Chondrin,  414 
Chondroitin,  50,  413 
Chondroitin-sulphuric  acid,  51,  413 
in  cartilage,  413 
isolation  of,  414 
properties  of,  413 
Chondromucoid,  414 

isolation  of,  414 
Chondrosin,  51,  413 
Choroid,  410 

Chromogen  of  adrenal  glands,  469 
Chromophanes,  410 
Chyle,  analysis  of,  369 
Chymosin,  143 

estimation  of,  145 


INDEX. 


489 


Chymosin  in  the  gastric  juice,  143 

in  the  pancreatic  juice,  153 

isolation  of,  144 

tests  for,  144 
Chymosinogen,  143 

estimation  of,  145 

test  for,  144 
Cilianic  acid,  168 
Citric  acid  in  milk,  449 
Clabber,  448 
Clupein,  54 

Coagulated  albumins,  61 
Coagulating  ferments,  119 
Coagulation  of  the  blood,  342 
rapidity  of,  347 
theories  of,  342 
Collagen,  59 
Colloidal  platinum,  20 
Colostrum,  450 

Compound  glycocolls  in  the  urine,  276 
Conalbumin,  458 
Conchiolin,  59 
Conjugate  glucuronates  in  the  urine,  274 

sulphates,  90 

estimation  of,  238 
in  the  urine,  267 
Connective  tissue,  embryonic,  411 
Copper  test  for  uric  acid,  256 
Cornea,  406 
Corpora  lutea.  455 
Cream,  440,  443 
a-Crotonic  acid,  92,  286 
Ci-usokreatinin,  108,  388 
Crystalline  lens,  407 

albumins  of,  407 
albumoid  of,  407 
crystallins  of,  407 
Crystallins,  407 
Cyan-haemoglobin,  360 
Cyanurin,  270 
Cyclopterin,  54 
Cystei'n,  88 

in  the  urine,  293 
Cysteinic  acid,  88,  170 
Cystin,  88 

estimation  of,  295 

in  the  kidney's,  438 

in  the  liver,  436 

in  the  urine,  293 

isolation  of,  295 

preparation  of,  296 

properties  of,  294 

transformation  to  taurin,  170 
Cvstinuria    associated    with    diaminuria, 

"293 
Cytosin,  99 

DAMALIC  acid,  283 
Damaluric  acid,  283 
Dare's  hsemometer,  359 

method  of  estimating  the  alkalinity 
of  blood,  333 
Dehydrocholalic  acid,  108 


Dehydrocholeic  acid,  169 
Denaturization,  31,  32 
Denniges'  test  tor  uric  acid,  256 
Dentin,  417 
Derived  albumins,  61 
Desamidoalbumin,  42 
Descemet's  membrane,  406 
Desoxycholalie    acid,  169 
Deutero-albumoses,  62,  187,  201 
Dextrins,  74 

in  the  urine,  305 
Dextrose.     See  Glucose. 
Diabetes,  298 
Diacetic  acid,  92,  286 

in  the  urine,  286,  287 
tests  for,  287 
Dialuric  acid,  101,  106,  253 
Diamido-acids,  82 
Diamido-nitrogen,  82 
Diamins  in  the  urine,  322 

isolation  of,  322 
Diaminuria,  322 
Diastases,  117 
Diethylene  diamin,  453 
Differential  density  method  of  estimating 

•sugar,  303 
Digestion,  182 

analysis  of  products  of,  198 

gastric,  185 

products  of,  198 

of  albuminoids,  192 

of  albumins,  185 

of  carbohvdrates,  182 

of  fats,  196 

of  proteids,  191 

tryptic,  189 

products  of,  199 
Digestive  fluids,  120 

glands,  436 
Di-oxv-phenyl-acetic  acid   in   the  urine, 

279 
Disaccharides,  70 
Diureids,  105 
Dry-pancreas,  152 
Ductless  glands,  466 
Dulcite,  66 
Dunlop's   method   of    estimating   oxalic 

acid,  261 
Dyslysin,  168 

EAR,  the,  410 
Egg-albumins,  457 
Eggs,  455 

albnmen  of,  455 
albumins  of,  457 
fats  of,  462 
incubation  of,  463 
lecithins  of,  463 
lipochromes  of,  462 
ovomucoid  in,  459 
ovovitellin  in,  461 
shell  of,  455 
yolk  of,  457 


490 


INDEX. 


Ehrlich's  diuzo  reaction,  316 
Eliistin,  t)U 
Elastoidin,  GO 
Elastoses,  02 
Eleidin  granules,  424 
Eiiiydin,  4()0 
Enamel  of  teeth,  417 
Eneephalin,  40.S 
Enteric  juice,  154 

amount  of,  15n 

enterokinase  in,  156 

erepsin  in,  156 

ferments  of,  155 

prosecretin  in,  157 

secretin  in,  157 
Enterokinase,  156 
Enzymes.     See  Ferments. 
Eosinophilic  crystalloids,  29 
Epiguanin,  103 
Epinephrin,  Abel's,  470 
Episarcin,  103 
Erepsin,  193 
Erythrodextrin,  123,  183 
Esbach's  reagent,  313 
Ethylene-succinic  acid,  82 
Ethylenimin,  453 
Ethyl  sulphide  in  the  urine,  292 
Euglobulin,  338 
Excretin  in  the  feces,  226 
Extractives  of  the  nerve-tissue,  404 
of  the  blood,  348 
of  the  liver,  436 
of  the  lymph,  368 
of  the  milk,  449 
of  the  thyroid  gland,  469 
Eye,  406 

"TAT,  76 

I      analysis  of,  420 

animal,  418 

bacterial  decomposition  of,  219 

chemistry  of,  76 

digestion  of,  196 

estimation  of,  in  the  milk,  448 

melting-point  of,  420 

of  birds'  egg,  462 

of  the  blood,  348 

of  the  liver,  435 

of  the  lympli,  367 

of  the  milk,  447 

of  the  muscle-tissue,  395 

of  the  urine,  320 

origin  of,  76 

in  milk,  447 

significance  of,  422 

synthesis  of,  in  plants,  26 
Fatty"  acids,  <)] 

estimaticm  of,  in  the  urine,  284 
isolation  of,  from  the  urine,  284 

degeneration,  435 

infiltration,  435 

tissue  (see  also  Adipose  tissue),  418 
Feces,  222 


Feces,  amount  of,  222 

bacteria  in,  223 

chemical  composition  of,  223 

color  of,  222 

consistence  of,  222 

crystals  in,  223 

excretin  in,  226 

form  of,  222 

hydrobilirubin  in,  225 

macroscopical  constituents  of,  223 

microscopical  constituents  of,  223 

odor  of,  222 

reaction  of,  223 

serolin  in,  226 

stercobilin  in,  226 

stercorin  in,  226 
Feliling's  method  of  estimating  sugar,  302 

solution,  299 

test  for  sugar,  299 
Fellic  acid,  169 
Fermentation,  acetic,  216 

alcoholic,  216 

butyric,  216 

lactic,  216 

method  of  estimating  sugar,  304 

tests  for  sugar,  300 
Ferments,  20,  ill 

amylolytic,  117 

chemical  composition,  115 

classification,  116 

coagulating,  119 

general  properties,  of,  113 
reactions  of,  115 

inverting,  117 

lipolytic,  117 

mode  of  action,  116 

of  the  adrenal  glands,  471 

of  the  enteric  juice,  155 

of  the  gastric  juice,  136 

of  the  liver,  434 

of  the  lymph,  369 

of  the  muscle-tissue,  378 

of  the  pancreatic  juice,  149 

of  the  urine,  321 

organized,  112 

oxidizing,  118 

proteolytic,  117 

reducing,  119 

reversible  action  of,  115 

tissue,  114 

which  cause  the  cleavage  of  urea,  117 

which  cause  the  cleavage  of  gluco- 
sides,  118 

which  cause  the  cleavage  of  nucle- 
inic  acids,  118 

which  split  off  carbon  dioxide,  118 

which  transform  amido-acids  to  am- 
ides, 117 
Ferratin,  433 
Ferri-albnminic  acid,  433 
Ferrocyanide  test  for  albumin,  310 
Fertilization  of  eggs,  463 
Fibrin,  61,  345 


INDEX. 


491 


Fibrin,  estimation  of,  346 
formation  of,  345 
isolation  of,  345 
l)roperties  of,  345 
test  for,  in  tlie  urine,  312 
Fibrin-ferment,  342 

chemical  nature  of,  343 
isolation  of,  344 
properties  of,  344 
Fibrin  of  llenle,  452 
Fibrinogen,  337 

isolation  of,  from  the  blood,  337 
properties  of,  337 
Fibrinoglobulin,  342 
Fibroin,  59 

Fibrous  tissue,  elastic,  412 
reticulated,  412 
white,  411 
vellow,  412 
Fish-scales,  418 
Fleischl's  hsemometer,  359 
Floridins,  353,  354 
Fluorides  in  the  urine,  237 
Folin's  method  of  estimating  urea,  249 

preformed      ammonia      in 

urine,  250 
uric  acid,  257 
Food-stufls  of  plants,  22 
Formic  acid,  91 

Freund's  method  of  estimating  the  acid- 
ity of  the  urine,  233 
Fructose.     See  Lrrvulone. 
Fuscin,  410 

GADUS  histon,  53 
Galactonic  acid,  70 
Galactose,  69 

Gallois'  test  for  inosit,  387 
Gases  of  the  blood,  335 

of  the  intestinal  contents,  219 
of  the  lymph,  369 
of  the  muscle-tissue,  395 
of  the  stomach  contents,  145 
of  the  urine,  321 
Gastric  contents,  acetic  acid  in,  135 
butyric  acid  in,  135 
lactic  acid  in,  134 
digestion,  185 
juice,  126 

acidity  of,  127 
amount  of,  126 
analysis  of,  127 
chemical  composition  of,  127 
ferments  of,  136 
gases  of,  1 45 

hydrochloric  acid  of,  129 
lactic  acid  in,  134 
pro-enzymes  of,  136 
sulphocvanides  in,  145 
Gelatin,  60 
Gelatoses,  62 

Gerhardt's  test  for  diacetic  acid,  288 
for  urobilin,  315 


Giacosa's  pigment,  270 
Glands,  adrenal,  46'J 

digestive,  436 

ductless,  466 

gastric,  126 

intestinal,  154 

lymph,  437 

mammary,  438 

reproductive,  451 

salivary,  120 
Globin,  53,  354 

isolation  of,  354 
Globulinoses,  62 
Globulins,  49 
Glucite,  66 
Glucoalbumins,  50 
Glucoalbumose,  201 
Glucocyamidin,  108 
Glucocyamin,  108 
Glucolysis,  348 

Glucolytic  ferment  of  Lepine,  348 
Gluconic  acid,  67,  275 
Glucoproteids,  46,  50 
Glucosamin,  45,  50,  68 
Glucose,  70 

estimation  of,  in  the  urine,  302 

in  the  blood,  348 

in  the  liver,  435 

in  the  muscle-tissue,  383 

in  the  urine,  297 

tests  for,  299 
Glucosides  in  nerve-tissue,  400 

synthesis  of,  in  plants,  25 
Glucosuria,  297 
Glucuron,  275 

Glucuronates,  conjugate,  274 
Glucuronic  acid,  274 

in  the  blood,  348 
in  the  urine,  274 
origin  of,  274,  275 
properties  of,  274 
tests  for,  275 
Glutamin,  209 
Glutaminic  acid,  85,  209 
isolation  of,  209 
Glutaric  acid,  93 
Glutin,  59 
Glutinoids,  59 
Glutokvrin,  190 
Glutolin,  348 
Glycerin-phosphoric  acid,  78 

in  the  urine,  321 
Glycocholic  acid,  87,  164 
isolation  of,  165 
Glvcocoll,  172,  210 

isolation  of,  172,  211,  278 

relation  to  hippuric  acid,  211,  27i 

test  for,  211 
Glycocolls,  compound,  276 
Glycogen,  74,  434 

estimation  of,  383,  435 

formation  of,  379 

in  the  blood,  348 


492 


INDEX. 


Glycogen  in  the  liver,  434 

in  tlie  mnscle-t issue,  .':)79 

isolation  of,  382 

properties  of",  434 

signiticance  of,  380 
Glycolic  acid,  \)'l 
Glycoluric  acid,  105 
Glycosuric  acid,  280 
Gmelin's  test,  175 
Govvers'  luemoglobinonieter,  359 
Granulose,  73 
Guanidin,  1U3 
Guanidin-butvric  acid,  83 
Guanin,  102, 'l 03,  393 

in  muscle-tissue,  393 

isolation  of,  391 

tests  for,  393 
Guano-biliary  acid,  1 65 
Guanylic  acid,  96,  436 

isolation  of,  436 
Gunning's  test  for  acetone,  289 
Giinzburg's  test  for  hydrochloric  acid,  131 

H.EMATIN,  354,  355 
in  the  urine,  317 

isolation  of,  357 
Htematinic  acids,  356,  363 
Hsematochlorine,  465 
Hifimatogen,  431,  461 
Hsematoidin,  363 
Hisniatoporphyrin,  22,  354,  362 

in  the  urine,  317 

preparation  of,  362 
Hsemin,  356 

preparation  of,  357 
Hfemoalkalimeter,  333 
Hseniochromogen,  354 

isolation  of,  354 
Hfemocyanin,  364 
Haemoglobin,  352 

amount  of,  356 

chemical  nature  of,  355 

isolation  of,  355 
Haemoglobinometer  of  Gowers,  359 
Haemoglobins,  59 
Hsemolymph,  364 
Hsemometer  of  Dare,  359 

of  Fleischl,  359 
Hammei-schlag's  method  of  determining 

the  specific  gravity  of  blood,  331 
Haptogenic  membrane  of  Ascherson,  439 
Hehner-Seeman's    method   of  estimating 

organic  acids,  136 
Helicoproteids,  56 
Heller's  test  for  albumin,  35 
for  blood,  317 

urrhodin,  270 
Hemi-albumose,  43 
Hemicelluloses,  75 
Hemipeptone,  43 
Henle's  fibrin,  452 
Hepatin,  431 
Hetero-albumose,  62,  186,  200 


Heterolysis,  114 
Heteroxanthin,  102 
Plexdbioses,  71 
llexou-bases,  54,  82 

in  antipeptone,  189 
Hexoses,  66 

Hippomelanin,  320,  425 
Hijipuric  acid,  87,  276 

estimation  of,  278 

isolation  of,  278 

of  the  urine,  276 

origin  of,  276 

properties  of,  277 

svnthesis  of,  278 
Histidin,  40,  48.  84 
Histons,  52,  350 

test  for,  in  the  urine,  312 
Histon-plasma,  347 
llistdzyme,  118,  277 
Ilotlhiann's  test  for  tyrosin,  207 
Homocerebrin,  402 
Homogentisinic  acid  in  the  urine,  281 

isolation  of,  282 
Hopkin's  method  of  estimating  uric  acid, 

256 
Hoppe-Seylei-'s  double  x)ipette,  359 

test  for  xanthin,  392 
Hupi)ert's  test,  176 
Hvalins,  52 
HValogens,  52,  418 
Hyalomucoid,  408 
Hyaloplasm,  326 
Hydantoic  acid,  105 
Hydantoin,  105 
Hydrations  in  animal  life,  19 
Hydrazons,  67 

Hydrobilirubin  in  the  feces,  225 
Hydrocele  fluid,  analysis  of,  370 
Hydrochloric  acid,  129 

combined,  132 

free,  129    _ 

of  gastric  juice,  129 

origin,  129 

quantitative  estimation  of  132 

secretion  of,  129 

significance  of,  130 

tests  for,  131 
Hydrocinnamic  acid,  94 
Hydrogen  ])eroxide  in  the  urine,  237 

sulphide  in  the  urine,  321 
Hydroparacumaric  acid,  93 

in  the  intestinal  contents,  219 

in  the  urine,  279 

isolation  of,  225 
Hydrocpiinon,  94 

in  the  urine,  267 
Hydurilic  acid,  105 
Hyocholalic  acid,  169 
Hyoglycocholic  acid,  165 
Hyolaurocholic  acid,  166 
Hvpobromite  method  of  estimating  urea, 

248 
Hypoxanthin,  102,  392 


INDEX. 


493 


Hypoxanthin  in  muscle-tissue,  392 

isolation  of,  391 
Hypoxanthylic  acid,  96 

TCIITHYDIN,  460 
i     Ichtbin,  460 
Ichthulin,  56 
Ichthulinic  acid,  462 
Ichthylepidin,  59 
Ilasvay's  reagent   125 
Incubation  of  eggs,  463 
Indican,  animal,  9U 

estimation  of,  272 
in  tlie  urine,  269 
origin  of,  269 
properties  of,  270 
tests  for,  271 
vegetable,  90 
Indiglucin,  90 
Indigo-blue,  90,  216,  270 
Indigo-purpurin,  270 
Indigo-white,  90 
Indirubin,  251 
Indol,  90,  216 

isolation  of,  224 
tests  for,  217 
Indoxyl,  90,  270 
Indoxyl red,  270 
Indoxyl  sulphate  (see  also  Indican),  90, 

269 
Inosinic  acid,  96 

in  nuiscle-tissue,  394 
Inosit  in  muscle-tissue,  387 
in  the  urine,  282 
isolation  of,  387 
tests  for,  387 
Intestinal  putrefaction,  215,  216 
Inulin,  74 
Invertases,  117    ' 
Invertins,  117 
Iron  albuminate,  432 
in  the  liver,  181 
Tsobilianic  acid,  168 
Isocholesterin,  80 
Isomaltose,  72,  123,  183 
Ivory,  417 

TAFFE'S  test  for  indican,  271 
»l  for  kreatinin,  265 

Jecorin  in  nerve-tissue,  405 

KATALASES,  118 
Katha?moglobin,  361 
Kaulosterin,  80 

Kelling's  test  for  lactic  acid,  134 
Kerasin.     See  Ilmnocercbrin. 
Keratin  in  the  skin,  424 
Keratinoses,  62 
Keratins,  52 
Ketoses,  66 
Kidneys,  438 

Kjeldahl  method  of  estimating  nitrogen, 
250 


Kinase,  pancreatic,  378,  437 

Knapp's  method  of  estimating  sugar,  302 

Knop-lliifner  method  of  estimating  urea, 

248 
Kornein,  59 
Kossell's  hypothesis,  44 
Kossler-Penny's    method    of    estimating 

urea,  230 
Kornein,  59 
Kossel's  hypothesis,  44 
Kossler-Penny's    method    of    estimating 

phenols,  268 
Kreatin,  108,  388 

in  muscle-tissue,  388 

in  the  urine,  263 

isolation  of,  390 

origin  of,  388 

properties  of,  389 

relation  to  kreatinin,  264,  389 
Kreatinic  leucomains,  108 
Kreatinin,  108,  388 

estimation  of,  265 

in  muscle-tissue,  388 

in  the  urine,  263 

isolation  of,  265 

origin  of,  264 

properties  of,  264 

relation  to  kreatin,  264,  389 

synthesis  of,  265 

tests  for,  265 
Kreatins,  108 
Kiihne's  amphopeptone,  186 

antipeptone,  189 
Kynurenic  acid,  283 
Kynuric  acid,  283 
Kynurin,  283 

LABORATOKY  exercises,  472 
Lactalbumin,  445 
estimation  of,  447 
isolation  of,  445 
Lactases,  117 
Lactic  acid,  92 

estimation  of,   in   the   stomach 

contents,  135 
in  the  muscle-tissue,  386 
in  the  stomach  contents,  134 
in  the  urine,  290 
isolation  of,  from  muscle-tissue, 

386 
origin  of,  in  nuiscle-tissue,  383 
properties  of,  386 
significance  of,  383 
tests  for,  134 
Lactoglobulin,  446 
estimation  of,  447 
isolation  of,  446 
Lactose,  72,  448 

estimation  of,  448 
in  the  milk,  448 
in  the  urine,  304 
isolation  of,  304,  448 
Lsevulinic  acid,  68,  99 


494 


ISDEX. 


Lfevulose,  69 

in  the  urine,  305 
Laiose,  oOo 
Lanolin,  .SO 
Luurinie  acid,  -147 
Lecitlialbumins,  79 
Lecithins,  78 

digestion  of,  197 

in  birds'  eggs,  463 

in  muscle-tissue,  368 

in  nerve-tissue,  403 

in  the  urine,  320 

isolation  of,  403,  463 
Legal's  test  for  acetone,  289 
Legumin,  56 

Leichenwax.     See  Adipoccre. 
Leo's  method  of  estimating  hydrochloric 
acid,  133 

sugar.     8ce  Laiose. 
Lepine's  glucolytic  ferment,  348 
Leucin,  85,  205 

in  the  urine,  291 

isolation  of,  208,  291 

tests  for.  206 
Lencinic  acid,  92 
Leucites,  21 
Leucocytes,  349 

chemical  composition  of,  350 
Leucomains,  109 

kreatinic,  108 

xanthinic,  103 
Leukonuclein,  350 
Leuko-urobilin,  223 
Lichenin,  74 

Lieben's  test  for  acetone,  289 
Liebermann's  albumin  reaction,  39 
Liebermann-Burckhard's  test  for  choles- 

terin,  180 
Lignin,  74 

Lilienfeld's  nucleohiston,  343,  349,  437 
V.  Limbeck's  method  of  estimating   the 

alkalinity  of  the  blood,  333 
Lipase  in  the  ga.stric  juice,  137 
Lipases,  222 
Lipochrin,  410 
Lipoch]-omes,  78,  462 
Lipochromic  pigments,  364,  462 
Lipuria,  320 
Lithofellic  acid,  169 
Lithuric  acid,  283 
Liver,  430 

albuminates  of,  431 

albumins  of,  431 

chemical  composition  of,  431 

extractives  of^  436 

fats  of,  435 

ferments  of,  434 

function  of,  430 

glucose  of,  435 

glycogen  of,  434 

nucleins  of,  432 
Living  matter,  chemical  changes  in,  18 
forces  at  work  in,  17 


Living  matter  general  composition  of,  17 
Lota-histon,  53 

Lowy's  methed   of  estimating  the  alka- 
linity of  the  blood,  333 
Ludwig-Salkowski  method  of  estimating 
uric  acid,  257 
of  estimating  xanthin-bases,  260 
Luteins,  the,  of  bird  eggs,  462 
Lymph,  365 

amount  of,  366 

analyses  of,  367,  369 

chemical  composition  of,  367 

general  properties  of,  366 
Lymphagogues,  366 
Lvmph-glands,  437 
Lysin,  40,  48,  84 
Lysuric  acid,  84 

MALONYI^UEEA,  105 
Maltase  in  the  pancreatic  juice,  153 
in  the  saliva,  124 
Maltases,  117 

Maltodextrin,  74 
Malto.se,  72,  183 

in  the  urine,  305 
Mammary  glands,  438 
Mann  ides,  synthesis  of,  in  plants,  25 
Mannite,  25,  66 
Mannitides.     See  3Linnides. 
Mannonic  acid,  67 
Manno-saccharinic  acid,  67 
Mannose,  66 
Meconium,  226 
Melanin.s,  319,  425 

in  the  skin,  425 

in  the  urine,  319 
Melanogen,  319 
Melanoidins,  425 
Mcmbranin,  52,  406 
Mesoporphyrin,  22,  363 
Mesoxalic  acid,  107 
Messinger-Muppert  method  of  estimating 

acetone,  290 
Metalbumin,  368 
Methsemoglobin,  361 

sulphide,  362 
]\Ietha^mog]oh)iniemia,  361 
Mcthvl-dibutvl-acetic  acid,  426 
Metliyl-glycocoU,  108 
Mothvl-guanidin,  108 
Methyl-hydantoin,  105,  108 

in  muscle-ti.ssue,  389 
Methvl-hvdantoinic  acid,  87 
MethVl-u'racil,  98,  101 
Mett's  method  of  estimating  pepsin,  142 
Milk,  439 

albumins  of,  443 

amount  of,  441 

analysis  of,  443 

casein  in,  443 

chemical  composition  of,  442 

citric  acid  in,  44'.t 

coagulation  of,  439,  445 


INDEX. 


495 


Milk,  extractives  of,  449 

fats  in,  447 

ferments  in,  450 

general  characteristics  of,  439 

lactalbumin  in,  445 

lactoglobulin  in,  446 

lactose  in,  448 

reaction  of,  441 

skimmed,  441,  443 

S2:)eciiic  gravity  of,  441 
Millon's  reaction,  38 
Mineral  ash,  estimation  of,  in  the  urine, 

237 
Molisch's  test,  39 
Mono-amido-acids,  40,  85,  205 

nitrogen,  47 
Monosaccharides,  66 
Mono-nreids,  105 

Morner  and  Sjoqvist's  method  of  estimat- 
ing hydrochloric  acid,  132 
of  estimating  urea,  248 

test  for  tyrosin,  208 
Mucin,  biliary,  161 

salivary,  124 
Mucinic  acid,  67,  70 
Mucinogen,  124 
Mucinoids,  50 
Mucins,  50 
Mucoids,  50 

corneal,  406 
Mucous  tissue,  411 
Murexid,  107 

test,  256 
Muse,  429 

Muscle-albumins,  significance  of,  376 
Muscle-pigments,  379 
Muscle-plasma,  373 
Muscle-serum,  374 
Muscle-stroma,  379 
Muscle-tissue,  372 

albumins  of,  373 

analyses  of,  372 

carnin  in,  394 

fat  in,  395 

ferments  of,  378 

gases  in,  395 

glucose  in,  383 

glycogen  in,  379 

guanin  in,  393 

hypoxanthin  in,  392 

inosinic  acid  in,  394 

inosit  in,  387 

involuntaiy,  396 

kreatin  in,  388 

kreatinin  in,  388 

lactic  acid  in,  383 

myogen  in,  374 

myosin  in,  375 

nitrogenous  extractives  of,  388 

nucleins  of,  377 

phospho-carnic  acid  in,  377 

pigments  of,  379 

plasma  of,  373 


Muscle-tissue  serum,  374 

stroma  of,  373,  379 

xanthin  in,  392 

xanthin-bases  in,  390 
Myelin,  79,  400 
Myelin-bodies,  400 
Myo-albumose,  377 
Myogen,  isolation  of,  374 

properties  of,  374 
Myogen-fibrin,  insoluble,  374,  375 

soluble,  374,  375 
Myoglobulin,  377 
Myoproteid,  377 
Myosin,  375 

isolation  of,  375 

properties  of,  375 
Myosin-tibrin,  37(3 
Myosinogen,  377 
Myosinoses,  62 
Myricin,  77 

Myristinic  acid,  180,  447 
in  the  bile,  ISO 
Myrosin,  118 
Myxoedema,  466 

NEOSSIN,  52 
Nerve-tissue,  397 
albumins  in,  398 
cerebrin  in,  401 
cholesterin  in,  404 
encephalin  in,  403 
extractives  of,  404 
general     chemical    composition 

of,  397 
homocerebrin  in,  402 
jecorin  in,  405 
lecithins  in,  403 
myelins  in,  400 
neuridin  in,  404 
neurokeratin  in,  399 
protagon  in,  400 
Neuridin  in  nerve-tissue,  404 
Neurin,  79 
Neurokeratin,  399 
Neutral  oxygen,  19 
sulphur,  292 
estimation  of,  296 
Nitric  acid  test  for  albumin,  308 
for  xanthin,  392 
oxide  hpemoglobin,  360 
Nitrites  in  the  saliva,  125 
test  for,  125 
in  the  urine,  239 
test  for,  239 
Nitrogen,  estimation  of,  250 
Nitrogenous  equilibrium,  243 
Nuclear  nucleins,  58 
Nucleases,  118 
Nucleinic  acids,  57,  95 

bases,  101,  259,  390 
Nuclein  platelets  of  the  blood,  351 
Nucleins,  58 

digestion  of,  191 


496 


INDEX. 


Nucleins,  isolation  of,  432 

of  tlie  liver,  432 
Nucleo-albumins,  50 

test  for,  in  the  urine,  310 
Kueleo-glucoproteid    of    the     mammary 
gland,  438 

of  the  pancreas,  43G 
Nucleo-histon,  343,  349,  437 

isolation  of,  349 

jiroperties  of,  350 
Nueleoproteids,  58 

of  the  mnscle-tissue,  377 
Nvlander's  test  for  sugai-,  299 

OBERMAYER'S  test  for  indican,  271 
Oleic  acid,  93 
Olein,  77 
Onuphin,  52 
Oocyanin,  456 
Oorhodein,  455 
Orcin  test,  386 

Organic  acids  in  stomach-contents,  esti- 
mation of,  130 
Ornithin,  82,  83,  87 
Ornithuric  acid,  84,  87,  279 
Osazons,  67 
Osons,  07 
Ossein,  415 
Ovalbumin,  457,  458 
isolation  of,  458 
Ovaries,  455 
Ovomucoid,  459 

isolation  of,  459 
Ovovitellin,  401 

isolation  of,  462 
properties  of,  461 
Ovum,  455 
Oxalate-plasma,  336 
Oxalic  acid,  93,  107 

estimation  of,  261 
in  the  urine,  260 
origin  of,  260 
Oxaluric  acid,  105 

in  the  urine,  260 
origin  of,  260 
Oxidations  in  animal  life,  19 
Oxy-acids,  aromatic,  279 
in  the  urine,  279 
isolation  of,  279 
tests  for,  279 
Oxy-amygdalic  acid  in  the  urine,  280 
i3-oxybutyric  acid.  92,  285 
estimation  of,  286 
relation  to  acetone,  286 
to  ra-crotonic  acid,  286 
to  diacetic  acid,  286 
tests  for,  286 
Oxydases,  118 

in  the  liver,  434 
in  the  saliva,  124 
Oxygenases,  118 
Oxyhfpmocyanin,  364 
Oxyhsemoglobin,  353,  355 


Oxyha'nioglobin,  estimation  of,  359 

isolation  of,  357 

))roperties  of,  358 
Oxy-hydroparacumaric  acid,  93 
Oxyi)]ienyl-ethy]anun,  89 
Oxyneurin,  79 
Oxyjiiperidin,  83 
Oxyproteinic  acid,  292 

PALMITIC  acid,  77,  91 
Palmitin,  77 
Pancreas,  436 

Kiihne's  dry,  152 
Pancreatic  juice,  146 

amount  of,  147 
chemical  composition  of,  148 
ferments  of,  149 
general  properties  of,  147 
significance  of,  140 
specific  gravity  of,  148 
zymogens  of,  149 
Paralbumin,  368 
Parabanic  acid,  103,  107 
Paracasein,  444 
Paracholesterin,  80 
Paracresol,  89 

in  the  urine,  267 
Paraglobulin,  338 
Parahiston,  53 
Paralactic  acid  in  the  urine,  290 

isolation  of,  290 
Paralbumin,  368 
Paranucleins,  56 
Para-oxy-benzoic  acid,  94 
Para-phenyl-acetic  acid,  88 
in  the  urine,  279 
Pani-oxy-phenyl-glycolic  acid,  94 

in  the  m'ine,  279 
Para-oxy-phenyl-lactic  acid,  93 

in  the  urine,  279 
Para-oxy-phenyl-i)ropionic  acid,  88,  94 

in  the  urine,  279 
Paraxanthin,  102 
Pekelharing's  method  of  isolating  pepsin, 

141 
Penta-methylene-diamin,  84,  322 
Pentoses,  70 

in  the  urine,  300 
tests  for,  306 
Pepsin,  139 

estimation  of,  142 
isolation  of,  141 
properties  of,  139 
Pepsinic  acid,  140 
Pepsinogen,  142 

estimation  of,  143 
test  for,  143 
Peptic  digestion,  products  of,  198 
Peptoids,  64,  186 
Pejttomelanin,  204,  425 
Peptone-plasma,  330 
Peiitones,  03,  187,  202,  203 
Pericardial  fluid,  analysis  of,  369 


INDEX. 


497 


Peritoneal  effusions,  analysis  of,  370 
Peroxydases,  118 
Pettenkofer's  test,  163 
Phenaceturic  acid,  87,  95,  279 
isolation  of,  279 
origin  of,  279 
properties  of,  279 
Phenol,  89 

estimation  of,  in  the  urine,  268 

in  the  intestinal  contents,  218 

in  the  urine,  267 

tests  for,  218 
Phenyl-acetic  acid,  94 

in  the  intestinal  contents,  219 
isolation  of,  225 
Phenyl-alanin,  89,  95 
Phenyl-ethylainin,  89 
Phenylhydrazin-test  for  sugar,  300 
Phenyl-propionic  acid,  94,  95 

in  the  intestinal  contents,  219 
isolation  of,  225 
Phlebin,  351 
Phloretin,  25 
Phloridzin,  25 

PhlorogUicin  test  for  pentoses,  307 
Phosphates,  estimation  of,  in  the  urine,  238 
Phospho-earnic  acid,  377 
Phosphoglobulins,  56 
Photo-methgemoglobin,  361 
Phyllocyanic  acid,  21 
Phylloporphyrin,  22,  363 
Phylloxanthin,  21 
Phymatorhusin,  319,  425 
Phytosterin,  80 
Pigments  of  the  bile,  172 

of  the  blood,  352 

of  the  muscle-tissue,  379 

of  the  skin,  425 

of  the  urine,  313 

respiratory,  353 
Pine-apple  test  for  butyric  acid,  135 
Piperazin,  453 
Piria's  test  for  tvrosin,  207 
Placenta,  465 
Plaques,  351 
Plasma,  albumose-,  336 

blood,  330,  336 

histon-,  347 

muscle-,  373 

oxalate-,  336 

peptone-,  336 

salt-,  336 
Plasminic  acid,  98 
Plasteins,  196 
Plastin,  327 
Platnei-'s  bile,  162 
Pleural  efiusions,  analyses  of,  370 
Plosz'  urorubrin,  270 
Polarimetric  test  for  glucose,  301 
Polypeptides,  Fischer's,  42 
Polysaccharides,  72 
Pro-enzymes,  111 
Propionic  acid,  91 

32 


Prosecretin,  157 
Protagon,  400 

isolation  of,  401 

properties  of,  400 
Protamins,  44,  54 

in  the  spermatozoa,  454 
Proteids,  57 

digestion  of,  191 
Proteinochromes,  212 
Proteins,  28 
Proteases,  117 
Proteoses,  62 
Prothrombin,  342 
Proto-albumose,  43,  62,  186,  200 
Protones,  64 
Protophyllin,  24 
Pseudoglobulin,  338 
Pseudomucin,  45 
Pseudonucleins,  56 
Pseudopepsin,  137 
Ptomains,  109 

acyclic,  109 

cyclic,  110 

in  the  intestinal  contents,  219 

in  the  urine,  322 
Ptyalin,  122 

action  of,  123 

chemical  nature  of,  124 

isolation  of,  123 
Purin,  100 

bases,  101 
Pus,  analysis  of,  371 

Putrefaction,  analysis  of  products  of,  224 
Putrescin,  83 

in  the  urine,  322 
Pyogenin,  400 
Pyosin,  400 
Pyrimidin  derivatives  of    the  nucleinic 

acids,  99 
Pyrocatechin,  94 

in  the  urine,  267 
Pyrocatechin-reaction  of  adrenal  glands, 

469 
Pyrocatechuic  acid,  280 
Pyrocholesteric  acid,  169 

REDUCTASES,  119 
Rennin.     See  Chymosin. 
Reproductive  glands,  451 
Resorption  of  albumins,  193 

of  carbohydrates,  184 

of  fots,  196 
Reticulin,  59,  412,  437 
Retina,  408 

analysis  of,  409 

chromophanes  in,  410 

rhodopsin  in,  409 
Reversible  action  of  ferments,  115 
Rhamnose,  70,  306 
Rhodophane,  410 
Rhodopsin,  409 
Rigor  mortis,  376,  383 
Rosenbach's  reaction,  271 


498 


INDEX. 


SACCHAEINIC  at'id,  67,  274 
Sacchaio-lactonic  acid,  67 
Saccharose,  71 
Salaiiiandriii,  429 
Salicin,  425 
Sali.LTcnin,  25 
Saliva,  120 

amount  of,  120 

analysis  of,  122 

chemical  composition  of,  121 

chorda-,  121 

digestive  importance  of,  124 

extractives  in,  126 

ferments  in,  124 

gases  in,  126 

general  characteristics  of,  120 

mineral  constituents  of,  126 

mucin  in,  124 

nitrites  in,  125 

paralytic,  121 

ptyalin  in,  122 

secretion  of,  120 

sulphocyanides  in,  125 

sympathetic,  121 
Salivary  glands,  120,  436 
Salkovvski's  method  of  estimating  oxalic 
acid,  262 

test  for  cholesterin,  180 
Salkowski-Volhard  method  of  estimating 

chlorides,  237 
Salniin,  54 
Salmon,  53 

vSalmonucleinic  acid,  96 
Salt-plasma,  336 
Saponification,  78 
Sarcin  (see  also  Hijpoxanthin) ,  102 

test  for,  392 
Sarcylic  acid,  96 
Scherer's  red  pigment  of  the  urine,  279 

test  for  inosit,  387 
for  leucin,  206 
for  tyrosin,  207 
Schiff's  test  for  uric  acid,  256 
Schmiedeberg's  histenzyme,  118,  277 
Schunk's  indirubin,  270 
Sclerotic,  406 
Scombrin,  54 
Scombron,  56 
Scvllite,  387 
Sebum.  428 
Secretin,  157 
Semen,  452 

general  properties  of,  452 
Sericin,  59 

Serolin  in  the  feces,  226 
Serum  of  the  blood,  341 

composition  of,  341 
Serura-albumin,  339 

forms  of,  339 

in  the  urine,  307 

isolation  of,  340 

of  the  blood,  339 

test  for,  310 


Serum-globulin,  337 

in  the  urine,  307 

isolation  of,  337 

ju-operties  of,  337 

test  for,  310 
Siderosis,  432 
Siegfried's  carniferrin,  377 

peptones,  187,  190 
Silicates  in  the  urine,  237 
Silurin,  54 
Skatol,  89,  217 

isolation  of,  224 

tests  for,  218 
Skatol-araido-acetic  acid,  41,  89,  212 
Skatol-carbonic  acid,  90,  217,  273 
isolation  of,  224 
test  for,  273 
Skatoxyl,  90 

sulphate  in  the  urine,  272 

test  for,  273 
Skeletins,  59,  418 
Skin,  424 

elei'din  granules  in,  424 

keratin  in,  424 
Smegma,  429 
Smith's  test,  176 
Soaps,  78 
Solanidin,  25 
Solanin,  25 
Sorbite,  66 

Spectroscopic  test  for  bilirubin,  176 
Spermanucleinic  acid,  95 
Spermatic  fluid,  453 
Spermatin,  453 
Spermatozoa,  452,  454 

analysis  of,  454 

chemical  composition  of,  454 

protarains  in,  454 
Spermin,  452,  453 

pliosphate  of,  452 
Spirographin,  52 
Spleen,  437 

arginin  in  the,  438 
Spongin,  59 
Spongioplasm,  326 
Starches,  73 
Steapsin,  152 
Stearic  acid,  93 
Stearin,  77 
Stercobilin  in  the  feces,  225 

relation  to  urobilin,  225 
Stercorin  in  the  feces,  226 
Stoke's  reagent,  354 
Stools  (see  also  Feces),  222 

acholic,  222 
Sturin,  54 
Succinic  acid,  93 

Sulphates,  estimation  of,  in  the  urine,  238 
Sulphocyanides  in  the  gastric  juice,  145 

in  the  saliva,  125 

in  the  urine,  292 

test  for,  125 
Sulphur,  acid,  of  the  urine,  292 


INDEX. 


499 


Sulphur,  neutral,  of  the  urine,  292 
Sulpluir-test,  39 
Supporting  tissues,  411 
Suprarenin,  v.  Fiirth's,  470 
Sweat,  426  _ 

analysis  of,  428 

chemical  composition  of,  428 

gases  of,  428 

general  characteristics  of,  427 

significance  of,  426 
Synalbumose,  186  (foot-note),  201 
Synaptase,  118 
Synovial  fluid,  371 
Synovin,  371 
Syntonin,  62,  186 

TARTRONIC  acid,  106 
Taurin,  87,  170_ 

in  muscle-tissue,  388 
isolation  of,  171 
relation  to  cystin,  294 
Taurocarbaminic  acid,  87 
in  the  urine,  293 
Taurocholic  acid,  87,  166 
isolation  of,  167 
Teeth,  417 
Testicles,  451 

Tetra-hydro-dioxypyridin,  83 
Tetra-methylene-diamin,  83 
Tlieobromin,  103 
Theophyllin,  103 
Thioalbumose,  201 
Thiosulphates  in  the  urine,  292 
Thrombin,  342 
Thrombosin,  343 
Thrombosin-calcium,  343 
Thymin,  98,  101 
Thyminic  acid,  97 
Thymo-nucleinic  acid,  96 
Thymus  gland,  437 
Thyreoglobulin,  467 
Thvreo-nucleo-proteid,  468 
ThVroid  gland,  466 
Thyroiodine,  466 
Tollens'  orcin  test,  306 

phloroglucin  test,  307 
Topfer's   method    of    estimating    hydro- 
chloric acid,  132 
Topfer's  test  for  hydrochloric  acid,  131, 

132 
Triglycerides,  77 
Trioxy-phenyl-propionic     acid     in     the 

urine,  279 
Triticonnucleinic  acid,  96 
Trypsin,  150 

action  of,  151 
isolation  of,  152 
test  for,  151 
Trypsinogen,  150 
Tryptic  digestion,  189 

products  of,  199 
Tryptophan,  89,  211 
tests  for,  212 


Tuberculosamin,  54 

Tuuicin,  418 

Ty rosin,  40,  42,  88,  206 

in  the  urine,  291 

isolation  of,  207,  291 

tests  for,  207 
Tyrosin-hydantoinic  acid,  87 

UFFELMANN'S  test  for  lactic  acid, 
134 
Umikoff's  reaction,  449 
Uracil,  100 
Uramic  acids,  87 
Uramido-benzoic  acid,  87 
Urases,  117 
Urea,  estimation  of,  248 

formation  of,  86,  240 

in  the  blood,  348 

in  the  urine,  240 

isolation  of,  from  the  urine,  247 

origin  of,  240 

properties  of,  245 

synthesis  of,  247 

tests  for,  245 
Urea-nitrate,  245 
Urea-oxalate,  246 
Ureids,  105 

Uric  acid,  101,  104,  105,  252 
estimation  of,  256 
in  the  urine,  252 
isolation  of,  256 
origin  of,  252 
properties  of,  255 
tests  for,  256 
Urine,  227 

acetone  in,  286,  288 

acidity  of,  233 

albumins  in,  307 

allantoin  in,  262 

ammoniacal  decomposition  of,  231 

amount  of,  229 

aromatic  constituents  of,  266 
oxy-acids  of,  279 

carbohydrates  of,  297 

chemical  composition  of,  234 

chlorides  in,  237 

c  hoi  ester  in  in,  320 

color  of,  228 

compound  glycocolls  in,  276 

conjugate  glucuronates  in,  274 
sulphates  in,  267 

cystei'n  in,  293 

cystin  in,  293 

dextrin  in,  306 

diacetic  acid-dn,  287 

fats  in,  320 

fatty  acids  in,  284 

ferments  in,  321 

gases  in,  321 

general  characteristics  of,  227 

glucose  in,  297 

hippuric  acid  in,  276 

homogentisinic  acid  in,  281 


500 


INDEX. 


Urine,  indican  in,  269 

inorj^anic  constituents  of,  234 

inot<it  in,  282 

kreatinin  in,  263 

kynurenic  acid  in,  283 

lactic  acid,  290 

lactose  in,  304 

kevulose  in,  305 

lecithin  in,  320 

leucin  in,  291 

litlmiic  acid  in,  2S3 

mineral  ash,  237 

maltose  in,  305 

neutral  sulphur  in,  292 

nitrates  in,  239 

nitrogenous  constituents  of,  240 

odor  of,  229 

organic  constituents  of,  239 

omithuric  acid  in,  279 

oxalic  acid  in,  270 

oxaluric  acid  in,  260 

/3-oxybutyric  acid  in,  285 

paralactic  acid  in,  290 

pentoses  in,  306 

phenaceturic  acid  in,  279 

phenols  in,  267 

phosphates  in,  238 

pigments  in,  313 

ptomains  in  322 

reaction  of,  230 

skatol-carbonic  acid  in,  273 

skatoxyl  sulphate  in,  272 

specific  gravity  of,  230 

sulphates  in,  238 

ty rosin  in,  291 

urea  in,  240 

uric  acid  in,  252 

urocaninic  acid  in,  283 

urohsematin  in,  271 

uroleucinic  acid  in,  280 

urorosein  in,  273 

volatile  fatty  acids  in,  284 

xanthin-bases  in,  259 
Urobilin,  isolation  of,  316 

Jaffe's,  315 

MacMunn's,  313 

tests  for,  315 
Urocaninic  acid,  283 
Urochloralic  acid,  275 
Urochrome,  313 

isolation  of,  314 
Urocyanin,  270 
Uroerythrin,  315 

isolation  of.  315 
Uroferric  acid,  292 
T'rofuscohfe matin,  318 
I  roglaucin,  270 
rroha?matin,  270,  271 

tests  for,  271 
Uroleucinic  acid  in  the  urine,  282 
Uroproteic  acid,  292 
Urorosein,  test  for,  273 
Uroroseinogen,  273 


Urorubin,  270 
Urorubroha'inatin,  318 
Uroxanthinic  acid,  280 
Urrhddin,  270 
Urrliodinic  acid,  280 
Ursocholeic  acid,  171 
Uterine  milk,  451 

YALEEIANIC  acid,  91 
V       Vitellins,  56 
Yitellolutein,  463 
Vitellorubin,  463 
Vitelloses,  62 
Vitreous  body,  408 

analysis  of,  408 

^TTANG'S  method  of   estimating  indi- 
VV       can,  272 

Weidel's  test  for  xanthin,  392 
Welckei-'s  method  of  estimating  the  total 

amount  of  blood,  332 
Weyl's  test  for  kreatinin,  265 
AVharton's  jellv,  411 
Whev,  acid,  448 

sweet,  440,  443 
"Witch's  milk,  451 

V.  Wittich's  method  of  isolating  pepsin, 
141 

XAXTHIX,  102,  259 
in  muscle-tissue,  392 

in  urine,  259 

isolation  of,  391 

properties  of,  392 

tests  for  392, 
Xanthin-bases,  101,  259,  390 

estimation  of,  260 

in  muscle-tissue,  390 

isolation  of,  391 

of  the  urine,  259 

origin  of,  259 
Xanthinic  leucomains,  103 
Xanthokreatinin,  108,  388 
Xanthophane,  410 
Xanthoproteic  reaction,  38 
Xanthopsin,  409 
Xanthvlic  acid,  96 
Xylose",  70,  306 

YEAST-XITLEIXIC  acid,  96 
Yolk  of  bird's  eggs,  459 
albumins  of,  461 
analysis  of,  461 
fats  of,  462 

hajmatogen  in,  431,  461 
lipochromes  of,  462 
ovovitellin  in,  461 
white,  459 
Yolk  platelets,  29 

ZYMASE,  112 
Zymogens,  1 1 1 
Zymolysin,  156 


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ESSIG  (CHARLES  J.).  PROSTHETIC  DENTISTRY.  Second  edition.  See 
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GREENE  (WILLIAM  H,).  MEDICAL  CHEMISTRY.  12mo,,  310  pages,  with 
74  illustrations.     Cloth,  $1.75. 

GRINDON  (JOSEPH),  A  POCKET  TEXT-BOOK  OF  SKIN  DISEASES. 
12mo.  of  367  pages,  with  39  illustrations.  Cloth,  $2.00;  flexible  leather,  $2.50,  net. 
Lea's  Series  of  Pocket  Text-Books.     Page  12. 


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GROSS  (SAMUEL  D.).  THE  URINARY  BLADDER,  THE  PROSTATE 
GLAND  AND  THE  URETHRA.  Third  edition,  revised  by  Samuel  W.  Grosp, 
M.  D.      Octavo  of  574  pages,  with  170  illustrations.     Cloth,  $4.50. 

GUENTHER  (A.  E.  AND  T.  C).  ^^V  EPITOME  OF  PHYSIOLOGY.  12mo., 
225  pages,  illustrated.    Cloth,  $1.00,  net.    Lea' s  Sei  ies  of  Medical  Epitomes.     See  page  10. 

HABERSHON  (S.  0.).  DISEASES  OF  THE  ABDOMEN.  Second  American  from 
the  third  English  edition.     Octavo,  554  pages,  with  11  engravings.     Cloth,  $3.50. 

HALE  (HENRY  E.).  AN  EPITOME  OF  ANATOMY.  Vlrno.,  389  pages,  71 
engravings.     Cloth,  $1.00,  ne<.     Lea' s  Series  of  Medical  Epitomes^.     See  page  10. 

HALL  (WINTIELD  S.).  TEXT-BOOK  OF  PHYSIOLOGY.  Octavo,  672  pages, 
with  343  engravings  and  6  colored  plates.     Cloth,  $4.00,  ne<;    leather,  $5.00,  7ie^ 

HAMILTON  (ALLAN  McLANE).  NERVOUS  DISEASES.  Second  and  revised 
edition.     Octavo,  598  pages,  with  72  engravings.     Cloth,  $4. 

HAMILTON  (MILDRED  M.).  A  POCKET  TEXT-BOOK  OF  MASSAGE. 
12m(),  about  400  pages,  illustrated.  Preparing.  Lea's  Series  of  Pocket  Text-Books.  See 
page  12. 

HARDAWAY  (W.  A.).  MANUAL  OF  SKIN  DISEASES.  Second  edition. 
12mo.,  560  pages  with  40  illustrations  and  2  colored  plates.     Cloth,  $2.25,  7iet. 

HARE  ( HOB  ART  AMORY ) .    A  TEXT-B  OOK  OF  PR  A  OTIC  A  L  THERA  PE  U- 

TICS,  with  Special  Keference  to  the  Application  of  Remedial  Measures  to  Disease  and 
their  Employment  upon  a  Rational  Basis.  >sinth  revised  edition.  Octavo,  851  pages, 
105  engravings,  4  colored  plates.  Cloth,  $4.00,  net ;  leather,  $5.00,  net;  half  morocco, 
$5.50,  net- 

PRACTICAL  DIAGNOSIS.  The  Use  of  Symptoms  in  the  Diagnosis  of  Dis- 
ease. Fifth  edition,  revised  and  enlarged.  Octavo,  727  pages,  with  236  engravings, 
and  25  full-page  plates.     Cloth,  $5.00  ;  leather,  $6.00  ;  half  morocco,  $6.50,  net. 


Editor.     A  SYSTEM  OF  PRACTICAL  THERAPEUTICS.     By  American 

and  Foreign  Authors.  Second  edition.  In  three  large  octavo  volumes  containing  2593 
pages,  with  457  engravings  and  26  full-page  plates.  Price  per  volume,  cloth,  $5.00, 
net;  leather,  $6.00,  net;  half  morocco,  $7.00,  net.  For  sale  by  subscription  only.  Full 
prospectus  free  on  application  to  the  publishers. 

ON  THE  MEDICAL  COMPLICATIONS  AND  SEQUELS  OF  TYPHOID 


FEVER.    Octavo,  276  pages,  21  engravings,  and  2  full-page  plates.     Cloth,  $2.40,  net. 

HARRINGTON    (CHARLES).      A  TREATISE   ON  PRACTICAL  HYGIENE. 

Second  edition.     Handsome  octavo  of  755  pages,  with  113  engravings  and  12  plates  in 
colors  and  monochrome.     Cloth,  $4.25,  net. 

HARTSHORNE  (HENRY).  A  CONSPECTUS  OF  THE  MEDICAL  SCIENCES. 
Comprising  Manuals  of  Anatomy,  Physiology,  Chemistry,  Materia  Medica,  Practice  of 
Medicine,  Surgery  and  Obstetrics.  Second  edition.  12mo.,  1028  pages,  with  477  illustra- 
tions.    Cloth,  $4.25;  leather,  $5. 

HAYDEN  (JAMES  R.).  A  POCKET  TEXT-BOOK  OF  VENEREAL  DIS- 
EASES. Third  edition.  In  one  12mo.  volume  of  304  pages,  with  66  engravings. 
Cloth,  $1.75,  net.  ;  Flexible  red  leather,  $2.25,  net.  Lea's  Series  of  Pocket  Text-Books. 
See  page  12. 

HAYEM  (GEORGES)  AND  HARE  (H.  A.).  PHYSICAL  AND  NATURAL 
THERAPEUTICS.  Heat,  Electricity,  Climate  and  Mineral  Waters.  Edited  by 
H.  A.  Hare,  M.D.     Octavo,  414  pages,  with  113  engravings.     Cloth,  $3. 

HERMAN  (G.  ERNEST).  FIRST  LINES  IN  MIDWIFERY.  12mo.,  198  pages, 
with  80  engravings.     Cloth,  $1.25.     Student^  Series  of  Manuals.     See  page  15. 

HERMANN  (L. ).  EXPERIMENTAL  PHARMACOLOGY.  ^  A  Hand-Book  of  the 
Methods  for  Determining  the  Physiological  Actions  of  Drugs.  Translated  by  Egbert 
Meade  Smith,  M.D.     12mo.,  199  pages,  with  32  engravings.     Cloth,  $1.50. 


Philadelphia,  706,  708  and  710  Sansom  St. — New  York,  111  Fifth  Avenue. 


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HERRICK  (JAMES  B.).  A  HANDBOOK  OF  DIAGNOSIS.  In  one  handsome 
12mo.   volume  of  429  pages,  with  80  engravings  and  2  colored  plates.     Cloth,  $2.50. 

HERTER    (C.  A.).      LECTURES    ON    CHEMICAL    PA2H0L0GY.     In   one 

12  mo.  volume  of  454  pages.     Cloth,  §1.75,  net. 

HILL  (BERKELEY).    SYPHILIS  AND  LOCAL  CONTAGIOUS  DISORDERS. 

In  one  Svo.  volume  of  479  pages.     Cloth,  $3.25. 

HILLIER  (THOMAS).  A  HANDBOOK  OF  SKIN  DISEASES.  Second  edition. 
In  one  royal  12mo.  volume  of  353  pages,  with  two  plates.     Cloth,  |2.25. 

HIRST  (BARTON  C.)  AND  PIERSOL  (GEORGE  A.).  HUMAN  MONSTROS- 
ITIES. jVJagniticent  folio,  containing  220  pages  of  text  and  illustrated  with  123  engrav- 
ings and  39  large  photographic  plates  from  nature.     In  four  parts,  price  each,  $5. 

HOBLYN  (RICHARD  D.).  A  DICTIONARY  OF  THE  TERMS  USED  IN 
MEDICINE  AND  THE  COLLATERAL  SCIENCES.  Thirteenth  edition.  In 
one  12mo.  volume  of  845  pages.     Cloth,  $3.00,  net. 

HOLLIS'  EPITOME  OF  MEDICAL  DIA  GNOSIS.  Lea's  Series  of  Medical  Epitcmes. 
See  page  10. 

HOLMES  (TIMOTHY).  A  TREATISE  ON  SURGERY.  Fifth  edition.  Octavo, 
1008  pages,  with  428  engravings.     Cloth,  $6 ;  leather,  $7. 

A  SYSTEM  OF  SURGERY.     Edited  by  John  H.  Packard,  M.D.      In  three 


Svo.  volumes^,  3137  pages,  979  engravings,  13  plates.     Per  volume.  Cloth, 

HUNTINGTON  (GEORGE  S.).  ABDOMINAL  ANATOMY.  Imperial  quarto, 
590  pages,  including  300  full-rage  plates.     De  luxe  edition,  $10.C0,  net. 

HYDE  (JAMES  NEVINS)  AND  MONTGOMERY  (FRANK  H.).  A  PRAC- 
TICAL TREATISE  ON  DISEASES  OF  THE  SKIN.  Sixth  edition,  thoroughly 
revised.  Octavo,  832  pages,  with  107  engravings  and  27  full-page  plates,  9  of  which 
are  colored.     Cloth,  $4.£0,  net;  leather,  $5.50,  net;  half  morocco,  $6.00,  net. 

JACKSON  (GEORGE  THOMAS).     THE  READY-REFERENCE  HANDBOOK 

OF  DISEASES  OF  THE  SKIN.     Fourth   edition.     12mo.    volume  of    617   pages, 
with  82  engravings,  and  3  colored  plates.     Cloth,  $2.75,  net. 

JAMIESON  (W.  ALLAN).  DISEASES  OF  THE  SKIN.  Third  edition.  Octavo, 
656  pages,  with  1  engraving  and  9  double-page  chromo-lithographic  plates.     Cloth,  $6. 

JEWETT  (CHARLES).  ESSENTIALS  OF  OBSTETRICS.  Second  edition. 
12mo.,  385  pages,  with  80  engravings  and  5  colored  plates.     Cloth,     $2.25,  net. 

THE  PRACTICE  OF  OBSTETRICS.     By  American  Authors.    Second  edition. 

One    octavo   volume  of  775  pages,   with   445  engravings  in  black   and  colors,  and   35 
full-page  colored  plates.     Cloth,    |5.00  ;  leather,  $6.00;  half  morocco,  $6.50. 

JULER  (HENRY).  A  HANDBOOK  OF  OPHTHALMIC  SCIENCE  AND 
PRACTICE.  Third  edition.  Octavo,  733  pages,  with  190  engravings,  25  plates.  Test- 
types  and  Color-Blindness  Test.     Cloth,  $5.25,  net. 

KELLY    (A.O.J.)     A    MANUAL    OF    THE    PRACTICE    OF   MEDICINE. 

Octavo,  about  600  pages,  illustrated.     Prej.aring. 

KIEPE  (EDWARD  J.).  EPITOME  OF  MATERIA  MEDICA  AND  THERA- 
PEUTICS.    Lea' s  Series  of  Medical  Epitomes.     See  page  10. 

KING  (A.  F.  A.).  A  MANUAL  OF  OBSTETRICS.  Ninth  edition.  In  one 
12mo.  volume  of  629  pages,  with  275  illustrations.     Cloth,  $2.50,  net. 

KIRK  (EDWARD  C).  OPERATIVE  DENTISTRY.  Second  edition.  See 
American  Text-books  (f  Dentistry,  page  2. 

KLEIN  (E.K  ELEMENTS  OF  HISTOLOGY.  Fifth  edition.  In  one  pocket-size 
12mo.  volume  of  506  pages,  with  296  engravings.  Cloth,  $2.00,  net.  Students'  Series  of 
Manuals.     Page  15. 

KOPLIK  ( HENRY ) .  DISEASES  OF  INF  A  NC  Y  A  ND  CHIL  DHO  OD.  Octar  o, 
675  pages  with  169  engravings  and  32  plates  in  black  and  colors.  Cloth,  $5.00  ;  leather, 
$6.00,  net. 

Philadelphia,  706,  708  and  710  Sansom  St.— New  York,  111  Fifth  Avenue. 


10  LEA    BROTHERS    &     CO.'S    PUBLICATIONS. 

LANDIS  (HENRY  G.).  THE  MANAGEMENT  OF  LABOR.  In  one  handsome 
r2mo.  %-olume  of  329  pages,  with  28  illustrations.     Cloth,  $1.75. 

LEA  (HENRY  C).  CHAPTERS  FROM  THE  RELIGIOUS  HISTORY  OF 
SPAIN;  CENSORSHIP  OF  THE  PRESS;  MYSTICS  AND  ILLUMINATI ; 
THE  ENDEMONIADAS;  EL  SANTO  NINO  DE  LA  GUARDIA;  BRI- 
ANDA  DE  BARDAXL     In  one  12mo.  volume  of  522  pages.     Cloth.  |2.50. 


A  HISTORY  OF  AURICULAR  CONFESSION  AND  INDULGENCES 

IN  THE  LATIN  CHURCH.     In  three  octavo  volumes  of  about  500  pages  each. 
Per  volume,  cloth,  $3. 

THE  MO  RISC  OS  OF  SPAIN:  THEIR  CONVERSION  AND  EXPULSION. 


In  one  royal  12mo.  volume  of  about  425  pages.     Extra  cloth,  $2.25,  net. 

STUDIES   IN  CHURCH  HISTORY.      New    edition.      12mo ,    605   pages. 


Cloth,  $2.50. 


SUPERSTITION  AND  FORCE;  ESSAYS  ON  THE  WAGER  OF  LAW, 

THE    WAGER    OF  BATTLE,   THE   ORDEAL   AND    TORTURE.     Fourth 
edition,  thoroughly  revised.     In  one  royal  12mo.  volume  of  629  pages.     Cloth,  $2.75. 

LEA'S  SERIES  OF  MEDICAL  EPITOMES.     Edited  by  V.  C.  Pkdersen,  M.D 
Covering  the  entire  field  of  medicine  and  surgery  in  twenty  two  convenient  volumes  of 
about  250  pages  each,  amply  illustrated  and  wriiten  by  prominent  teachers  and  specialists. 
Compendious,  authoritative  and  modern.    Following  each  chapter  is  a  series  of  questions 
which  will  be  found  convenient  in  quizzing.     The  Series  is  constituted  as  follows  : 

Hale's  Anatomy.  Guenther's  Physiology.  McGlannan's  Inorganic  Chemistry  and 
Physics.  McGlannan's  Organic  and  Physiological  Chemistry.  Kiepe's  Materia  Medica 
and  Therapeutics.  Dayton's  Practice  of  Medicine.  Hollis's  Medical  Diagnosis.  Arneill's 
Clinical  Diagnosis  and  Urinalysis.  Nagel's  Nervous  and  Mental  Diseases.  Wathen's 
Histology.  iStenhouse's  Pathology.  Archinard's  Bacteriology  and  Microscopy.  Magee 
and  Johnson's  Surgery.  Ailing  and  Griffen  on  the  Eye  and  Ear.  Ferguson  on  the  Nose 
and  Throat.  Schmidt's  Genito-Urinary  and  Venereal  Diseases.  Schalek's  Dermatology. 
Pedersen  and  Parker's  Gynpecology.  Manton's  Obstetrics.  Tuley's  Pediatrics.  Dwight's 
Jurisprudence.  Dwight's  Toxicology.  For  separate  notices  see  under  various  authors' 
names. 

LEA'S  SERIES  OF  POCKET  TEXT-BOOKS.    See  page  12. 

LE  FEVRE  (EGBERT).  A  TEXT-BOOK  OF  PHYSICAL  DIAGNOSIS.  12mo., 
450  pages,  74  engravings,  12  plates.     Cloth,  $2.25,  net. 

LONG  (ELI  H.).  DENTAL  MATERIA  MEDICA  AND  THERAPEUTICS. 
12mo.,  321  pages,  6  engravings,  18  plates.     Cloth,  $3.00,  net. 

LOOMIS  (ALFRED  L.)  AND  THOMPSON  (W.  GILMAN),  Editors.  A  SYS- 
TEM  OF  PRACTICAL  MEDICINE.  In  Contributions  by  Various  American  Authors. 
In  four  very  handsome  octavo  volumes  of  about  900  pages  each,  fully  illustrated  in  black 
and  colors.  Per  volume,  cloth,  $5 ;  leather,  $6 ;  half  Morocco,  $7.  For  sale  by  sub- 
scription only.     Full  prospectus  free  on   application. 

LYMAN  (HENRY  M.).  THE  PRACTICE  OF  MEDICINE.  In  one  very  hand- 
some octavo  volume  of  925  pages  with  170  engravings.     Cloth,  $4.75;  leather,  $5.75. 

MAGEE  (M.  D.)  AND  JOHNSON  (WALLACE).  AN  EPITOME  OF  SUR- 
GERY. 12rao.,  about  300  pages,  with  loO  engravings.  Cloth,  $1.00,  net.  Lea's  Series 
of  Medical  Epifomea.     See  page  10. 

MAISCH  (JOHN  M.).  A  MANUAL  OF  ORGANIC  MATERIA  MEDICA. 
Seventh  edition,  thoroughly  revised  by  H.  C.  C.  Maisch,  Ph.G.,  Ph.D.  In  one  12mo. 
of  512  pages,  with  285  engravings.     Cloth,  $2.50,  net. 

MALSBARY  (GEO.  E.).  A  POCKET  TEXT-BOOK  OF  THEORY  AND 
PRACTICE  OF  MEDICINE.  12mo.  405  pages,  with  45  illustrations.  Cloth,  $1.75, 
net;  flexible  red  leather,  $2.25,  net.     Lea's  Series  of  Pocket  Text- Books.     Page  12. 


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LEA    BROTHERS    &     CO.' S    PUBLICATIONS.  11 

MANTON  (W.  P.).     AN  EPITOME   OF   OBSTETRICS.     12mo.,  265  pages,  82 

illustrations.     Cloth,  1 1.00,  Me<.     Lea' s  Series  of  Medical  Epitomes.     See  page  10. 

MANUALS.     See  Medical  Epitomes,  page  10  ;  Pocket  Text-Books,  page  12. 

MARSH  (HOWARD).  DISEASES  OF  THE  JOINTS.  In  one  12mo.  volume  of 
468  pages,  with  64  engravings  and  a  colored  plate.  Cloth,  $2.  See  Series  of  OlinvMl 
Manuals,  page  13. 

MARTIN  (EDWARD.)  SURGICAL  DIAGNOSIS.  One  12mo.  volume  of  400 
pages,  richly  illustrated.     Preparing. 

MARTIN  (WALTON)  AND  ROCKWELL  (W.  H.,  JR.).  A  POCKET  TEXT- 
BOOK OF  CHEMISTRY  AND  PHYSICS.  12mo.  366  pages,  with  137  illus- 
trations. Cloth,  $1.50,  net;  flexible  leather,  |2.00,  net.  Lea's  Series  of  Pocket  Text- 
Books.     Page  12. 

McGLANNAN  (A.).  AN  EPITOME  OF  INORGANIC  CHEMISTRY  AND 
PHYSICS.  12mo,  216  pages,  with  20  engravings.  Cloth,  fl.OO,  net.  Lea's  Series  of 
Medical  Epitomes.     See  page  10. 

AN  EPITOME  OF  ORGANIC  AND  PHYSIOLOGICAL  CHEMISTRY. 

12mo.,  246  pages,  with  9  engravings.    Cloth,  |1.00,  net    Lea's  Series  of  Medical  Epitomes. 
See  page  10. 

MEDICAL  EPITOME  SERIES.     See  Lea's  Series  of  Medical  Epitomes,  page  10. 

MEDICAL  NEWS  POCKET  FORMULARY.    See  page  1.    $1.50,  net. 

MITCHELL  (JOHN  K.).  REMOTE  CONSEQUENCES  OF  INJURIES  OF 
NERVES  AND  THEIR  TREATMENT.  12mo.,  239  pages,  12  illustrations.  Cloth, 
$1.75. 

MITCHELL  (S.  WEIR).  CLINICAL  LESSONS  ON  NERVOUS  DISEASES. 
12mo.,  299  pages,  with  17  engravings  and  2  colored  plates.     Cloth,  $2.50. 

MORROW  (PRINCE  A.).  SOCIAL  DISEASES  AND  MARRIAGE.  SOCIAL 
PROPHYLAXIS.     Octavo,  390  pages.     Cloth,  $3.00,  ne^     Just  ready. 

MUSSER  (JOHN  H.).  A  TREATISE  ON  MEDICAL  DIAGNOSIS,  for  Students 
and  Physicians.  New  (fifth)  edition,  thoroughly  revised  and  rewritten.  Octavo,  1205 
pages,  with  395  engravings,  and  63  full- page  colored  plates.  Cloth,  $6.50,  net;  leather, 
$7.50,  net;  half  morocco.  $8.00,  net. 

NAGEL  (J.  D.)  AN  EPITOME  OF  NERVOUS  AND  MENTAL  DISEASES. 
12mo.,  about  250  pages,  illusti-ated.  Shortly.  Lea's  Series  of  Medical  Epitomes.  See 
page  10. 

NATIONAL  DISPENSATORY.     See  StUle,  Maisch  &  Caspari,  page  14. 

NATIONAL    FORMULARY.      See  National  Dispensatory,  page  14. 

NATIONAL  MEDICAL  DICTIONARY.    See  BiUings,  page  3. 

NETTLESHIP  (E.).  DISEASES  OF  THE  EYE.  Sixth  American  from  sixth 
English  edition.  Thoroughly  revised.  12mo.,  562  pages,  with  192  engravings,  5  colored 
plates,  Test-types,  Formulae  and  Color-blindness  Test.     Cloth,  $2.25,  net. 

NICHOLS  (JOHN  B.)  AND  VALE  (F.  P.).  A  POCKET  TEXT-BOOK  OF 
HISTOLOGY  AND  PATHOLOGY.  12mo.  of  459  pages,  with  213  illustrations. 
Cloth,  $1.75,  net;  flexible  leather,  $2.25,  net.    Lea's  Series  of  Pocket  Text-Books.    Page  12. 

NORRIS  (WM.  F.)  AND  OLIVER  (CHAS.  A.).  TEXT-BOOK  OF  OPHTHAL- 
MOLOGY. In  one  octavo  volume  of  641  pages,  with  357  engravings  and  5  colored 
plates.     Cloth,  $5 ;  leather,  $6. 

OWEN  (EDMUND).    SURGICAL  DISEASES  OF  CHILDREN.     In  one  12mo. 

volume  of  525  pages,  with  85  engravings  and  4  colored  plates.     Cloth,  $2.     See  Series  oj 
Clinical  Manuals,  page  13. 

PARK  (WILLIAM  H.).     BACTERIOLOGY  IN  MEDICINE  AND  SURGERY. 

12mo.,  688  pages,  87  engravings  in  black  and  colors,  2  colored  plates.     Cloth,  $3.00,  net. 


Philadelphia,  706,  708  and  710  Sansom  St— New  York,  111  Fifth  Avenue. 


12  LEA    BROTHERS    &     CO.'S    PUBLICATIONS. 

PARK  (ROSWELL),  Editor.  A  TREA  TTSE  ON  SURGERY,  by  American  Authors. 
For  Students  and  Practitionei's  of  Surgery  and  Medicine.  Third  edition.  In  one 
large  octavo  vohime  of  1408  pages,  with  692  engravings  and  64  plate.s.  Cloth,  $7.00; 
leather,  $8.00,  Hf^  Published  also  in  2  volumes:  Vol.  I.,  General  Surgery;  Vol.11., 
Special  or  Regional  Surgery.     Per  volume,  cloth,  $3.75,  vet;  leather,  $4.75,  net. 

PEDERSEN  AND  PARKER'S  EPITOME  OF  GYNECOLOGY.  Lea's  Series  M 
Mt  (ileal  Epitomes.     See  page  10. 

PEPPER  (A.  J.).  SURGICAL  PATHOLOG  Y.  In  one  12mo  volume  of  511  pages, 
with  81  engravings.     Cloth,  $2.     See  Studentis'  Series  of  Manuals,  page  15. 

PICK  (T.  PICKERING).  FRACTURES  AND  DISLOCATIONS.  In  one  12mo. 
volume  of  530  pages,  with  93  engravings.    Cloth,  $2.    See  Series  of  Clinical  Manuals,  p.  13. 

PLAYFAIR  (W.  S.).  THE  SCIENCE  AND  PRACTICE  OF  MIDWIFERY. 
Seventh  American  from  the  ^'inth  English  edition.  Octavo,  700  pages,  with  207  engrav- 
ings and  7  full  page  plates.     Cloth,  $3.75;  leather,  $4.75,  net. 

POCKET  FORMULARY.     Fifth  edition.     See  page  1. 

POCKET  TEXT-BOOK  SERIES  covers  the  entire  domain  of  medicine  in  eighteen 
volumes  of  350  to  525  pages  each,  written  by  teachers  in  leading  American  medical  col- 
leges. Issued  under  the  editorial  supervision  of  Bern  B.  Gallaudet,  AI.D.  ,  of  the  College 
of  Physicians  and  Surgeons,  New  York.  Thoroughly  modern  and  authoritative,  concise 
and  clear,  amply  illustrated  with  engravings  and  plates,  handsomely  printed  and 
bound.  The  series  is  constituted  as  follows:  Eockwell's  Anatomy;  Collins  &  Rock- 
well's Physiology;  Martin  &  Rockwell's  Chemistry  and  Physics;  Nichols  &  Vale's 
Histology  and  Pathology;  Schleif's  Materia  Medica  and  Therapeutics  ;  Malsbary's  Prac- 
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Eye,  Ear,  Nose  and  Throat  ;  Evans'  Obstetrics;  Crockett's  Gynecology  ;  Tuttle  on  Dis- 
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POLITZER  (ADAM).  A  TEXT-BOOK  OF  THE  DISEASES  OF  THE  EAR 
AND  ADJACENT  ORGANS.  Third  American  from  the  Fourth  German  edition. 
In  one  octavo  volume  of  896  pages,  with  346  engravings.     Cloth,  $7.50,  net. 

POSEY  (W.  C.)  AND  WRIGHT  (JONATHAN).  A  TREATISE  ON  THE 
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650  engravings  and  35  plates  in  black  and  colors.  Cloth,  $7.00;  leather,  $8.00,  net. 
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II.,  Wright  on  the  Nose,  Throat  and  Ear.     Cloth,  $3.50,  net. 

POTTS  (CHAS.  S.).  A  POCKET  TEXT-BOOK  OF  NERVOUS  AND 
MENTAL  DISEASES.  12mo.  of  455  pages,  with  88  illustrations.  Cloth,  $1.75,  net; 
flexible  leather,  $2.25,  net.     Lea's  Series  if  Pocket  Text-Books,  page  12. 

A    TEXT-BOOK   ON   MEDICINE  AND   SURGICAL  ELECTRICITY. 

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PROGRESSIVE  MEDICINE.    See  page  1.    Per  annum,  $9.00,  in  cloth  ;  $6.00  in  paper. 

PURDY  (CHARLES  W.).  BRIGHT'S  DISEASE  AND  ALLIED  AFFEC- 
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ings.    Cloth,  $2. 

PYE-SMITH  (PHILIP  H.).  DISEASES  OF  THE  SKIN.  In  one  12mo.  volume 
of  407  pages,  with  28  illustrations,  18  of  which  are  colored.     Cloth,  $2. 

RALFE  (CHARLES  H.).  CLINICAL  CHEMISTRY.  In  one  12mo.  volume  of 
314  pages,  with  16  engravings.     Cloth,  $1.50.     See  Students'  Series  of  Manuals,  ^age  14. 

REMSEN  (IRA).  THE  PRINCIPLES  OF  THEORETICAL  CHEMISTRY. 
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REYNOLDS  (EDWARD)  AND  NEWELL  (F.  S.).  MANUAL  OF  PR  A  CTICAL 
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NAL  SUROERY.      Octavo,  about  800  pages,  profusely  illustrated  with  engravings 
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ROBERTS  (JOHN  B.).     THE  PRINCIPLES  AND  PRACTICE  OF  MODERN 

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ROBERTS  ( SIR  WILLIAM ) .  A  PR  A  CTICA  L  TREA  TISE  ON  URINA  R  Y  AND 
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ROGER  (G.  H.).  INFECTIOUS  DISEASES.  Translated  bv  M.  S.  Gabriel,  M.D. 
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SCHAFER  ( EDWARD  A. ) .  THE  ESSENTIALS  OF  HISTOL  OGY,  DESCRIP- 
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volume  of  843  pages,  with  507  illustrations,  mostly  original.  Cloth,  $5.50,  net.  Just 
ready. 

WICKS  MAUD  (A.).  A  POCKET  TEXT-BOOK  OF  NURSING.  12mo.,  about 
400  pages,  illustrated.     Preparing.     Lea's  Series  of  Pocket  Text-Books.     Page  12. 

WILLIAMS  (DAWSON).  MEDICAL  DISEASES  OF  INFANCY  AND 
CHILDHOOD.  S  cond  edition  specially  revised  for  America  by  F.  S.  Churchill, 
A.M.,  M.D.     Octavo,  53  i  pages,  52  engravings  and  2  colored  plates.      Cloth,  $3.50,  net. 

WILSON  (ERASMUS).    A  SYSTEM  OF  HUMAN  ANATOMY.     Revised  edition, 

octavo,   616  pages,  with  397  engravings.     Cloth,  $4 ;  leather,  $5. 

WOOLSEY  (GEORGE).  APPLIED  SURGICAL  ANATOMY  REGIONALLY 
PRESENTED.  Octavo,  511  pa^es,  with  125  original  illustrations  in  black  and 
colors.     Cloth,  . "Jo. 00;  leather,  $6.00,  ?ie^ 

ZAPFFE  (FRED.  C.)  A  POCKET  TEXT-BOOK  OF  BACTERIOLOGY.  12mo., 
3.50  pages  with  14t  engravings  and  7  colored  plates.  Cloth,  $1.50  ;  flexible  leather, 
$2.00,  net.     Lea's  Series  of  Pocket  Text- Books.     Page  12. 


Philadelphia,  706,  708  and  710  Sansom  St— New  York,  111  Fifth  Avenue. 


QP519 
Simon 


Si5 
1904 


T -: ■> 


